Polypeptide and protein derivatives and process for their preparation

ABSTRACT

Protein and polypeptide derivatives and their salts are claimed characterized in that a protein or polypeptide is conjugated via an intermediate grouping containing at least one radical of the formula —C(R)═N— (or —N═C(R)—) or —CH(R)—NH— (or —NH—CH(R)—), wherein R is hydrogen or a hydrocarbon residue which may be substituted, with the same or a different protein or polypeptide, with a reporter group or a cytotoxic agent as well as a process for their preparation and the novel intermediates therefor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/089,051, filed Aug. 6, 1993, now abandoned, which is a continuationof application Ser. No. 07/866,262, filed Apr. 10, 1992, now abandoned,which is a continuation of application Ser. No. 07/742,159, filed Aug.1, 1991, now abandoned, which is a continuation of application Ser. No.07/506,545, filed Apr. 5, 1990, now abandoned, which is a continuationof application Ser. No. 07/380,738, filed Jul. 17, 1989, now abandoned,which is a continuation of application Ser. No. 07/220,196, filed Jul.18, 1988, now abandoned, which is a continuation of application Ser. No.07/043,530, filed Apr. 28, 1987, now abandoned.

TECHNICAL FIELD

This invention relates to novel polypeptide and protein derivatives inwhich polypeptides and proteins are conjugated by bridging molecules tothe same kind of polypeptides or proteins, other kinds of proteins orpolypeptides, reporter groups or cytotoxic agents.

BACKGROUND OF THE INVENTION

In the diagnosis of many forms of disease, as well as when following theeffects of treatment, it would often be desirable to use labelledproteins that bind to specific target structures in the body. Forexample, when diagnosing or treating cancer, it would be desirable to beable to detect both primary tumours and metastases using labelledtumour-specific antibodies. Many reports have appeared on the labellingof proteins and antibodies by random chemical attack on their sidechains. In such a process, most frequently, the side chains of thetyrosines are iodinated (Mach et al., Cancer Research 43, 5593-5600[1983]), or the side-chains of the lysines are acylated. In this lattercase the acylation is often by groups that chelate metals (e.g.Hnatowich et al., Science 220, 613-615 [1983]). Subsequently, thechelating groups can be used to bind radioactive metals. It has alsobeen suggested but not yet been satisfactorily tested to bind to suchmolecules paramagnetic ions for nuclear magnetic resonance (NMR) imaging(Brady et al., Magnetic Resonance in Medicine 1, 286 [1984]). Thelabelling of proteins, especially of antibodies, however, has so faralways been effected in a more or less random way.

Random substitutions on biological active proteins, for example randomsubstitutions on antibody molecules, can have a number of drawbacks:

1. If by chance a particularly reactive site were to lie in the activesite of the protein a substitution at this site would possiblyinactivate the protein, e.g. a particularly valuable monoclonal antibodymight be rendered totally useless if by chance a side chain particularlyreactive towards substitution were to lie in the antigen-binding site.The substitution would then inactivate the antibody.

2. Even when the active site of the protein (e.g. an antibody) escapesserious damage, a high number of substitutions on the protein—which maybe desirable, e.g. in order to have a high intensity in case ofradioactive labelling via chelating groups—might change itsphysico-chemical properties (e.g. solubility).

3. A random, multiple substituted product constitutes a heterogenousmixture of molecules with different properties, with attendant problemsof assuring constant properties from batch to batch.

SUMMARY OF THE INVENTION

The present invention relates to novel polypeptide and proteinderivatives, to a process for their preparation, to their use and tonovel intermediates therefor. The novel polypeptides and proteins of thepresent invention are, more specifically, polypeptides and proteinswhich are conjugated via an intermediate grouping containing at leastone radical of the formula —C(R)═N— (or —N═C(R)—) or —CH(R)—NH— (or—NH—CH(R)—), wherein R is hydrogen, an aliphatic, cycloaliphatic,aromatic or araliphatic hydrocarbon group which group may besubstituted, with themselves or each other, with a different polypeptideor protein or with a reporter group or a cytotoxic agent. Thesecompounds are obtained by condensation of two reactants one of which isan aldehyde (or acetalized aldehyde) or ketone the other being an aminocompound thus yielding a Schiff base or azomethine type compound which,if desired or necessary, can be stabilized in a further reaction, viz.by reduction of the —C(R)═N— (or —N═C(R)—) radical to a —CH(R)—NH— (or—NH—CH(R)—) radical.

DESCRIPTION OF THE INVENTION

The present invention in a major aspect makes use of the fact thatenzymes can direct bifunctional reagents with suitable reactive groupsat specific sites in polypeptides or proteins (e.g. antibodies). Thesesites are preferably the carboxyl terminus of the polypeptide chain,which is at least in terms of primary structure in most cases far fromthe active site of proteins. This is especially true for antibodymolecules where the carboxyl terminus is furthest away from theantigen-binding site. Therefore problem No. 1 mentioned above can beeliminated by the process of the present invention. The limitation ofthe substitution to a specific site such as the carboxyl terminus, willalso eliminate problems No. 2 and No. 3, above.

However, in a further aspect the present invention makes use of the factthat specific bifunctional reagents with suitable reactive groupspreferably or specifically react at non carboxy terminus sites of themolecule, viz. with specific side chains or the amino terminal aminogroup in a non-enzymatic reaction.

Examples of bifunctional reagents with suitable reactive groups arecompounds with an amino group at one end and with a formyl or aminogroup (preferably in protected form) at the other end, such as o-, m- orp-formylphenylalanine.

Therefore, the polypeptide and protein derivatives of the presentinvention can be prepared by a condensation reaction between an aldehydeor ketone and an amino compound to yield the desired derivative of theazomethine or Schiff base type and, if desired, subsequent reduction ofthe —C═N— radical (which is relatively labile in case one of thereaction partners is an amine and the product is a Schiff base) to forma corresponding derivative containing a —CH₂—NH— radical. The aminocompound can be an amine, an O-alkylated hydroxylamine or a hydrazide.In the case of an O-alkylated hydroxylamine reacting with a carbonylcompound (aldehyde or ketone) oximes are obtained containing a—C(R)═N—O— radical. Since such compounds are relatively stable nosubsequent reduction, albeit possible, is necessary to form acorresponding derivative containing a —CH(R)—NH—O— radical. In the caseof a hydrazide reacting with a carbonyl compound the reaction productwill contain a —C(R)═N—NH— radical which again is relatively stable andneeds no reduction to form a corresponding derivative containing a—CH(R)—NH—NH— radical.

The basic reaction scheme of which the present makes use is>C═O+H₂N—→>C═N—→>CH—NH—. In this scheme, one complementary group(carbonyl or amino) is placed at the N- or C-terminus of a protein orpolypeptide under mild conditions. To obtain specificity (discriminationbetween an attached amino group and lysine side chains of the protein orpolypeptide) a reactive amino group attached to a protein must be anaromatic one, i.e. must be directly attached to an aromatic group, suchas phenyl or it must be directly attached to —O— or to —NH—CO—, i.e. bean O-alkyl-hydroxylamine or a hydrazide, respectively.

If at least one of the reactive groups (carbonyl or amino group) of thereaction partners is aromatic, preferably if both are aromatic, it wasfound that the condensation reaction is rapid, and highly efficient evenat surprisingly low concentrations of reactants. The reactivitiesinvolved are sufficiently great to permit the attachment of, e.g. apolymeric chelating group to the specific site, which means that at thecost of a single modification at a specific site on the protein known tobe safe for this purpose, it is possible to introduce virtually as manyof the desired substituent groups as required for high radioactivity.This feature again permits to overcome problem No. 2, addressed above,since a high number of substitutions spread over the whole protein inorder to achieve a high enough intensity of labelling is no longerrequired.

For the reasons discussed above, it is usually preferable to have thegroup which is to participate in the condensation reaction to form aSchiff base type compound attached specifically, via enzymatic methods,to the carboxy terminus of the protein or polypeptide.

Under certain circumstances, however, it may be satisfactory andconvenient to form Schiff base links via groups introduced elsewhere andgroups introduced by other methods. Usually, but not always, suchmethods are less specific than the carboxy-terminal enzymatic method.Circumstances under which these other methods might be employed are:

(i) in cases where the carboxy-terminal region is important for functionor should not be altered for other reasons and

(ii) where particular properties of the protein or polypeptide, e.g. itspossessing a single or rather few side chain residues of an amino acidfor which specific chemical modification reagents exist or may bedesigned, can usefully be exploited.

Therefore, it is possible to combine group-specific chemicalmodification of protein or polypeptide side chains with subsequentcoupling to Schiff bases. In the context of the present specificationand claims the term “Schiff base” is meant to extend to all protein orpolypeptide derivatives exhibiting a >C═N— radical and thus alsoencompasses compounds, such as, oximes or hydrazones. A wide variety ofgroup-specific protein modification reagents are known which permit themodification, with various degrees of specificity, of the functionalgroups present in proteins. Furthermore, many examples exist where twoof the chemically reactive groups present in such reagents have beenincorporated into a single molecule to provide a bivalent reagent (seee.g. the Catalogue of the Pierce Chemical Co., the world's leadingmanufacturer of protein cross-linking reagents). So far, none of thesereagents have been used for Schiff base chemistry. It should be notedthat great advantage is to be made from combining, in the same molecule,a group capable of reacting with functional groups of proteins orpolypeptides and a group capable (after deprotection, if it is used inprotected form) of forming a Schiff base link with a complementary groupon another molecule, viz. a carbonyl or amino group. Such reagents arerepresented by the general formula R³—X—R¹, where R³ is a chemical groupwhich reacts with functional groups of proteins or polypeptides, X is abivalent organic group or may be absent but is preferably an aromaticradical directly adjacent to R¹ and must be an aromatic group or oxygendirectly adjacent to R¹ where R¹ is amino or protected amino and R¹ iscarbonyl, acetalised formyl (e.g. dimethoxy or diethoxy methyl), aminoor protected amino. Suitable amino protecting groups are those which arestable enough to withstand the attachment of R³ to the polypeptide orprotein, yet labile enough to be removed under conditions which do notirreversibly denature the polypeptide or protein. Many such groups areknown to the art, e.g., citraconyl, trifluoroacetyl, Boc, BPOC, MSC.Suitable groups for R³ are well known to the art (c.f., for example,Means, G. E. and Feeney, R. E. [1971] “Chemical Modification ofProteins”, Holden-Day, San Francisco): groups that react selectivelywith amino-groups are, e.g., active-esters such as hydroxysuccinimideesters, o-nitrophenyl esters, imidates or haloaromatics with a nucleusactivated to nucleophilic substitution; groups that react selectivelywith sulphhydryl-groups are, e.g., haloalkyls, activated disulphides,aziridines, activated vinyl compounds; groups that react selectivelywith guanido-groups are, e.g., alpha- or beta-dicarbonyls;aromatic-group selective reagents are, e.g., diazonium compounds;indole-group selective compounds are, e.g., aromatic sulphenyl halides,and carboxyl-group selective reagents are, e.g., diazoalkanes and aminocompounds in the presence of condensing reagents such as DCCI.

The polypeptide and protein derivatives of the present invention can berepresented by the formula

A—X—Z—X′—B  (I)

wherein

A is the residue of a protein or polypeptide;

B is the residue of a protein or polypeptide, of a reporter group or ofa cytotoxic agent;

X and X′ independently from each other are bivalent organic radicals ormay be absent;

Z is a bivalent radical selected from the group consisting of —C(R)═N—,—N═C(R)—, —CH(R)—NH—, —NH—CH(R)—, —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)— or —NH—CH(R)—Y—CH(R)—NH—, wherein R is hydrogen,an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group,which group may be substituted with the same or a different protein orpolypeptide, a reporter group or a cytotoxic agent, with at least onearomatic radical or oxygen adjacent to nitrogen and

Y is a bivalent organic group

and salts thereof.

The invention also extends to salts of the protein and polypeptidederivatives mentioned above, especially to metal salts thereof. Of mostinterest among the salts are the metal chelate complexes useful for invivo imaging.

Thus, the compounds of formula I are derivatives of (or modified)polypeptides or proteins. Typical polypeptides and proteins, theresidues of which are designated A and B in formula I, are on the onehand those occurring in nature and capable of being isolated from natureindependent from whether their structure and/or amino acid sequences andglycosylation pattern has already been identified or not and on theother hand those which have been or can be prepared synthetically orsemisynthetically in accordance with methods well-known in the art.Preferred compounds of formula I are derivatives of polypeptides andproteins of medical interest, among which, e.g., derivatives ofimmunoglobulins, especially antibodies of the IgG type. It will beappreciated that not only complete antibodies can be labelled orderivatised using the method of the present invention but also subunitsthereof which are still functional, such as F(ab′)₂ or Fab fragments.

Formula I comprises compounds of the following types:

A—X—C(R)═N—X′—B  (I-A′)

A—X—N═C(R)—X′—B  (I-A″)

A—X—CH(R)—NH—X′—B  (I-B′)

A—X—NH—CH(R)—X′—B  (I-B″)

A—X—C(R)═N—Y—N═C(R)—X′—B  (I-C′)

A—X—N═C(R)—Y—C(R)═N—X′—B  (I-C″)

A—X—CH(R)—NH—Y—NH—CH(R)—X′—B  (I-D′)

A—X—NH—CH(R)—Y—CH(R)—NH—X′—B  (I-D″)

wherein A, B, X, X′, R and Y are as defined above.

With R=hydrogen (i.e. with one of the reaction partners being analdehyde or a protected aldehyde) compounds of the following generalformulae are obtained:

A—X—CH═N—X′—B  (I-a′)

A—X—N═CH—X′—B  (I-a″)

A—X—CH₂—NH—X′—B  (I-b′)

A—X—NH—CH₂—X′—B  (I-b″)

 A—X—CH═N—Y—N═CH—X′—B  (I-c′)

A—X—N═CH—Y—CH═N—X′—B  (I-c″)

A—X—CH₂—NH—Y—NH—CH₂—X′—B  (I-d′)

A—X—NH—CH₂—Y—CH₂—NH—X′—B  (I-d″)

wherein A, B, X, X′ and Y are as defined above.

In the case where B represents a protein or polypeptide residue theseresidues may be different from or identical with A. In the first casehetero-dimers of proteins and polypeptides and in the second casehomo-dimers of proteins and polypeptides can be obtained.

B may alternatively represent the residue of a cytotoxic agent or areporter group. Cytotoxic agents in the present context are defined tocomprise all compounds generally summarized under this expression suchas cytostatics and toxins. Cytostatics of specific interest are thosechemo-therapeutically active compounds against cancer, i.e.cancerostatics (carcinostatics). The term “reporter group” is meant todefine compounds which are easily detectable by analytical means invitro and/or in vivo and which confer this property to compounds towhich they are bound. This term comprises, e.g., any organiccompounds/groups which are capable of binding strongly to metals(including and preferably radioactive metals). Especially preferredamong such reporter groups are metal chelating agents/groups (chelons),e.g., desferrioxamine or DTPA (systematic names see below). Apart frombeing a compound/group capable of being radioactively labelled, reportergroups may be fluorescent groups or groups capable of being monitored byNMR or ESR spectroscopy.

The groups X and X′ can be absent or represent bivalent radicals ofaliphatic, aromatic or araliphatic compounds and can be substituted. Xand X′ may be identical or can differ from each other, e.g., only onemay be present. Preferred groups X and X′ are aromatic radicals, e.g.,—NH—C₆H₄— or araliphatic radicals, e.g., —NH—CH₂—CH₂—C₆H₄—,—NH—CH(COOCH₃)—CH₂—C₆H₄— or —NH—CH(CONH₂)—CH₂—C₆H₄—, since the formingof the Schiff base type compounds is favoured in this case. Examples ofaliphatic groups X or X′ are —O—CH₂—CO—, —NH—CH₂—CO—,—NH—CH₂—CH₂—S—CH₂—. It is essential that in case there are aliphaticamino groups present in the protein or polypeptide molecule (whichlatter case generally happens, e.g., if lysine is present) that thearomatic group is adjacent to the N-atom of group Z of formula —N═CH— or—NH—CH₂—, which means that the Schiff base formation occurs via anaromatic amino group at the side of the protein or polypeptide. In casethe protein, polypeptide or the cytotoxic agent or reporter groupcontains already such an aromatic amino group through which the couplingcan be effected X and/or X′ will be absent. In this case the N-atom of Zoriginates from the starting protein, polypeptide, cytotoxic agent orthe reporter compound. The same applies with respect to an aromaticformyl function.

In order to reach highest specificity in the coupling reaction it ispreferred either to use aromatic aldehydes and aromatic amino compoundsso that in the Schiff base compounds obtained the radicals adjacent toboth the N- and the C-atom of the —CH═N— or —N═CH— group are aromaticgroups, preferably phenylene groups, or to use ketones andO-alkylhydroxylamines.

In case one of the reaction partners is a ketone it is preferable to useamino compounds which are stronger nucleophiles than arylamino compoundsand which are known to react rapidly, specifically and under mildconditions with carbonyl groups. Such amino compounds includesubstituted hydrazines (hydrazides) and O-substituted, preferablyO-alkylated, hydroxylamines, such as H₂N—O—CH₂—COOH. The stability atnon-extreme pH of hydrazones and oximes means that the reductionstep >C═N—X→>CH—NH—X, which is required when X is aryl, is notnecessary, albeit possible. In the case of a hydroxylamino compoundbeing used as nucleophile compound of formula I will be obtained whereinZ is a bivalent radical selected from the group consisting of —CH═N—O—,—O—N═C(R)—, —CH(R)—NH—O—, —O—NH—CH(R)—, —C(R)═N—O—Y—O—N═C(R)—,—O—N═C(R)—Y—C(R)═N—O—, —CH(R)—NH—O—Y—O—NH—CH(R)— and—O—NH—CH(R)—Y—CH(R)—NH—O—, with R and Y being as defined above.

Compounds of formulae I-C′, I-C″, I-D′ and I-D″ are obtained when adiamino compound of formula H₂N—Y—NH₂ or a dicarbonyl compound offormula OC(R)—Y—C(R)O is reacted with a carbonyl or an amino compoundrespectively. Y can be any bivalent organic group, i.e. an aliphatic,aromatic or araliphatic group. For obvious reasons simple molecules arepreferred. A most preferred aromatic Y group is phenylene while in caseof an aliphatic Y group this group has two O- or NH-radicals. It is alsoevident that although compounds of formulae I-C′, I-C″, I-D′ and I-D″can be prepared using methods well-known in the art wherein A and Band/or X and X′ are different, the preferred compounds of that type arethose wherein B is identical with A and X′ is identical with X(including the possibility that both latter groups are absent). Thussymmetric proteins or polypeptide dimers are obtainable which arecoupled almost specifically via a —C—N—Y—N—C— or a —N—C—Y—C—N— chain.

The compounds of formula I and their salts in accordance with theprocess of the present invention are obtained by condensing a compoundof formula

A—X—R¹  (II)

wherein

R¹ is —CO—R, acetalized formyl or amino and

A is a residue of a protein of polypeptide, X is a bivalent organicradical or may be absent, and R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group, which groupmay be substituted with the same or a different protein or polypeptide,a reporter group or a cytotoxic agent,

with a compound of formula

R²—X′—B  (III)

wherein

R² is amino in case R¹ in the compound II above is —CO—R or acetalizedformyl and is —CO—R or acetalized formyl in case R¹ in compound II aboveis amino and X′ is a bivalent organic radical or may be absent, B is aresidue of a protein or polypeptide, a reporter group or a cytotoxicagent, and R is hydrogen or an aliphatic, cycloaliphatic, aromatic oraraliphatic hydrocarbon group, which group may be substituted with thesame or a different protein or polypeptide, a reporter group or acytotoxic agent,

or condensing a compound of formula II above with a compound of formula

R²—Y—R²  (IV)

wherein

Y is as defined above and

R² is amino in case R¹ in the compound II above is —CO—R or acetalizedformyl and is —CO—R or acetalized formyl in case R¹ in compound II aboveis amino to form a Schiff base and, if desired, reducing the —C(R)═N— or—N═C(R)-radical(s) generated by the condensation to —CH(R)—NH— or—NH—CH(R)-radical(s) respectively and, if desired, forming a salt.

Thus either a carbonyl compound A—X—C(R)O, in case of R═H an aldehyde oran acetal thereof, preferably the methyl or ethyl acetal, is reactedwith an amino compound, preferably an aromatic amine H₂N—X′—B or anO-derivative of hydroxylamine, or an amino compound, preferably anaromatic amine A—X—NH₂ or an O-derivative of hydroxylamine, is reactedwith a carbonyl compound O(R)C—X′—B, in case of R═H an aldehyde or acorresponding acetal, preferably the methyl or ethyl acetal, to form theSchiff base.

If symmetric bisproteins or bispolypeptides are desired a carbonylcompound A—X—C(R)O or an acetal thereof, in case R═H, is reacted with adiamino compound H₂N—Y—NH₂, or an amino compound A—X—NH₂, preferably anaromatic amino compound or an O-derivative of hydroxylamine, is reactedwith a carbonyl compound O(R)C—Y—C(R)O or an acetal thereof, in caseR═H.

As follows from the definitions of A, B and Z above the amino orcarbonyl (or acetalized formyl) groups R¹ and R² in compounds offormulae II and III which participate in the formation of the Schiffbase type bond are connected to the residues A and/or B either via thebivalent organic group X and/or X′ respectively or may be part of theresidues A and B respectively, in which latter case X and/or X′ in theresulting compound of formula I are/is absent.

It should be noted that at least one of the reacting carbonyl and aminogroups is an aromatic group, viz. is directly connected to an aromaticgroup so that in a compound of formula I at least one aromatic group isdirectly adjacent to Z or that, alternatively, in case of an aliphaticcarbonyl compound the amino compound is a hydrazide or hydroxylaminoO-derivative.

Consequently, if in a compound A—X—R¹ (II) X has an aliphatic groupadjacent to R¹, the reactive carbonyl or amino function R² in thecompound of formula III must be an aromatic or araliphatic one, i.e. X′must have an aromatic group adjacent to R² or the amino function shouldbe derived from hydrazine or hydroxylamine, while if X has an aromaticgroup adjacent to R¹ or the amino function is derived from hydrazine orhydroxylamine, the reactive carbonyl or amino function, R² in thecompound of formula III may be adjacent either to an aromatic oraliphatic group, but aromatic is preferred.

The structure of the aliphatic or aromatic groups X′ and/or X is notcritical. The aromatic groups may be derived from a hydrocarbon or froma heterocycle; they are preferably derived from benzene, viz. either ofthem is or both are phenylene radicals which may be substituted. Theonly limitation with respect to the substituents of X′ and/or X is thatthey should not interfere with the reaction of the amino or carbonylgroup, i.e. should not react instead of the amino or carbonyl groups,should not be a sterical hindrance or should not deactivate the reactivegroups.

Compounds of the following general formulae are examples of subgroups ofcompounds of the general formula I

A—C(R)═N—X′—B  (I-E′)

A—X—C(R)═N—B  (I-F′)

A—C(R)═N—B  (I-G′)

A—N═C(R)—X′—B  (I-E″)

 A—X—N═C(R)—B  (I-F″)

A—N═C(R)—B  (I-G″)

A—C(R)—NH—X′—B  (I-H′)

A—X—C(R)—NH—B  (I-I′)

A—C(R)—NH—B  (I-K′)

A—NH—C(R)—X′—B  (I-H″)

A—X—NH—CH(R)—B  (I-I″)

and

A—NH—CH(R)—B  (I-K″).

With R=hydrogen (i.e. with one of the reaction partners being analdehyde or a protected aldehyde) compounds of the following subgroupsof general formula I are obtained:

A—CH═N—X′—B  (I-e′)

A—X—CH═N—B  (I-f′)

A—CH═N—B  (I-g′)

A—N═CH—X′—B  (I-e″)

A—X—N═CH—B  (I-f″)

A—N═CH—B  (I-g″)

A—CH₂—NH—X′—B  (I-h′)

A—X—CH—NH—B  (I-i′)

 A—CH₂—NH—B  (I-k′)

A—NH—CH₂—X′—B  (I-h″)

A—X—NH—CH—B  (I-i″)

and

A—NH—CH₂—B  (I-k″).

The condensation between compounds II and III can be carried out inaccordance with methods well-known in the art under mild conditions indilute solutions. The reaction the most intensively studied was that ofdes-Ala^(B30)-insulin-B29-formylanilide with m-aminobenzoyl-ferrioxamineB. The most generally useful ranges of conditions are describedimmediately below. However, as will be seen from the appended Examplesthe reaction conditions are easily and successfully applicable to otherreactants in spite of considerable differences in their nature.

The reaction can be carried out with good results at a pH range of3.5-5.5. Any suitable aqueous buffer can be used. The buffer wasnormally aqueous acetic acid (1%, v/v) adjusted to the desired valuewith NaOH solution. Insulin derivatives are poorly soluble at the upperend of this pH range, but the addition of solid urea overcame thisproblem without any detectable effect on coupling efficiency.Dimethylformamide could also be used as a solubilizing agent, at thecost of some slowing down of the coupling. Similar problems with otherprotein derivatives may be overcome in a similar way.

The concentration of the insulin-aldehyde derivative was usually between500 μM and 1 mM, that of the other reactant, m-aminobenzoyl ferrioxamineB, was usually between 500 μM and 2.5 mM. The reactants can be used inequimolar amounts or up to a multiple excess of one of the components.

Couplings can be carried out at ambient temperature. The half-time ofthese reactions, as judged by HPLC after quenching by dilution in acid,is of the order of one to two minutes. After 20-40 minutes yields aregenerally already at a maximum and the starting product is almostimperceptible.

The protein-carbonyl derivative can be used either as the free carbonylcompound or, especially in case of aromatic aldehyde, as an acetal,preferably as the methyl or ethyl acetal. The free compound stillcoupled efficiently after storage as a freeze-dried powder at roomtemperature for some months. In theory, the acetal-protected formsshould have been deprotected before coupling, but it proved possible totake advantage of their lability to acid below pH 6 (which is fargreater in case of aromatic acetals, than the lability of aliphaticacetals) and allow them to deprotect in the coupling mixture. If itturns out that at pH 5.5 no coupling occurs, the pH may be lowered. AtpH 3.5 there will most probably be no difference in the reaction speedbetween the acetal protected form and the free aldehyde.

When of appropriate structure the compounds of the Schiff base-typeobtained may be isolated and purified. It is well-known that Schiffbases are readily hydrolyzed and relatively unstable due to easycleavage of the —CH═N-bond. However, in some cases such lability may beof advantage and, therefore, explicitly desired. Schiff base-typecompounds obtained from two aromatic reactants (aromatic aldehyde groupand aromatic amino group) are more stable than those obtained with oneof the reactants being aliphatic. Oximes and hydrazones are more stablethan simple Schiff bases.

Therefore, it is generally desirable to stabilize the Schiff base-typecompounds. This is most conveniently done by reduction of the —CH═N-bondto a —CH₂—NH-bond and accomplished in a manner well-known in the artusing complex metal hydrides, preferably sodium cyanoborohydride orpyridine borane. Only a small excess of cyanoborohydride is necessaryand technical grade product can be used without disadvantage. Where ahigher purity is desirable it can be purified by precipitation fromacetonitrile by the method of Jentoft and Dearborn (J. Biol. Chem. 254,4359-4365 [1979]). Otherwise, pyridine borane may be used (Wong et al.,Anal. Biochem. 139, 58-67 [1984]).

The compounds of formula I (conjugates) obtained can easily betransformed into salts using methods well known in the art. In the caseof conjugates wherein B is the residue of a chelating agent (chelon),metal salts, especially salts with radioactive metals are the desiredend products useful as valuable tools in diagnosis and therapy. Everykind of radioactive salt can be obtained by simply mixing aprotein-chelon conjugate with an appropriate solution of a radioactivemetal and it is believed that the improved techniques for conjugation ofproteins provided by the present invention will lead to improvements inradio-immunoassay technique, histo- and cytochemistry, and in vivoimaging. Once the protein-chelon conjugates have been prepared, they canbe stored for long periods. It should subsequently be possible to labelthem whenever wished with alpha, beta, gamma, positron, and even neutronemitters under mild and strictly comparable conditions. The method willbe equally applicable to NMR imaging with paramagnetic ions serving ascontrast agents. If a gamma emitter is desired the protein may belabelled with ¹¹¹In (specific activity >5000 Ci/mmol) while a suitablepositron emitter is ⁶⁸Ga (max. theoretical specific activity 2.7×10⁷Ci/mmol).

The protein or polypeptide derivatives of formula II which are used asstarting materials in the coupling reaction of the present invention maybe prepared by reacting a protein or polypeptide with a suitablebifunctional reagent using methods well known in the art. It is easilypossible to, e.g. acylate side chain amino groups of proteins andpolypeptides with bifunctional reagents. In that case when an aliphaticor aromatic compound of formula R³—X—R¹ is used containing one function(R³) capable of reacting with a reactive group of a protein side chainand a second reactive group R¹, wherein R¹ as well as X are as definedabove, compounds of formula A—X—R¹ (II) are obtained wherein the proteinor polypeptide residue is connected via a side chain. While in such areaction, e.g., acylation will occur at several sites and a mixture ofdifferent compounds II will be obtained, it is preferable to usereaction conditions by which the point of attachment is limited to asingle selected region of the protein or polypeptide. A preferredselected region in connection with the present invention is the carboxyterminus of the protein or polypeptide.

In recent years proteolytic enzymes have already been used in thesynthesis of peptide bonds. This is possible because the enzymecatalyzed proteolysis is a reversible reaction. The method has beendescribed by several authors (see e.g. Jakubke et al., Angew. Chemie,Int. Ed. Engl., 24, 85-93 [1985]) and has been used alreadysuccessfully, e.g. in the preparation of human insulin (see e.g. Rose etal., Biochem. J. 211, 671-676 [1983]; European published patentapplication No. 87 238). Especially suitable enzymes useful in suchreverse proteolysis which can be used to prepare compounds which are thepreferred coupling reagents in the process of the present invention aretrypsin and carboxypeptidase Y. However, other enzymes can also be used,with the best reaction conditions being easily determined in somepreliminary experiments.

The general reaction can be described by the following equation

wherein A, X and R¹ are as defined above and the carboxyl group is theC-terminus of the molecule.

In specific examples the coupling of p-aminophenylalanine amide (withcarboxypeptidase Y) and of m-amino-benzaldehyde methyl and ethyl acetal(with trypsin) to the C-terminal region of the B-chain of insulin isdescribed below. These compounds are representations of bifunctionalmolecules of the general formula R³—X—R¹ which are especially useful inconnection with the present invention. In these and all followingreactions no protection whatever was needed for the protein's functionalgroups. Under the semi-aqueous conditions that have been chosen for thetrypsin-catalyzed reaction, synthesis is greatly favoured overhydrolysis. Only Lys^(B29) is affected and the final product wasdes-Ala^(B30)-insulin-B29-m-formylanilide.

Normally, carboxypeptidase Y progressively attacks the C-terminus ofproteins. Such a degradation can be carried out under conditions thatfavour synthesis at the same time as hydrolysis (Widmer et al. inPeptides 1980, 46-55 (editor K. Brunfeldt). Scriptor Kopenhagen [1981];Widmer et al. in Peptides 1982, 375-379 (eds. Blaha and Malon), W. deGruyter Berlin and New York [1983]). Under such circumstances, while amixture is obtained, it is one in which useful products predominate. Iflarge polypeptides and proteins are used the heterogeneity of the endproduct which may exist due to continuous degradation of the proteinfrom the C-terminus or perhaps of the enzyme's inserting more than onemolecule of compound R³—X—R¹, is, however, of minor importance since itremains restricted to a small region, viz. the C-terminus, of themolecule and will generally not be crucial to its activity.

The above coupling principle which can be extended to all proteins ofinterest is of specific interest in view of its applicability toimmunoglobulins, especially antibodies of the IgG type, and to fragmentsthereof, such as F(ab′)₂ or Fab. Trypsin produces Fab-like fragmentsanalogous to those made with papain, and the above equation applies tothe fixation of a unique site for conjugation at the C-terminus thereof,a region known to be far from the antigen-binding site. It cannot beexcluded that papain might also participate in such a coupling reactionand give directly the wanted derivatives of Fab fragments. Furthermore,carboxypeptidase Y will also introduce points of attachment (for theformation of Schiff base type, derivatives) at the C-termini of all thechains of IgG. F(ab′)₂, and Fab molecules, once again far from theantigen combining sites.

The feasibility of the carboxypeptidase Y approach has been studied inextenso. The possible range of conditions in the coupling ofp-amino-phenylalanine amide to insulin with carboxypeptidase Y wasexplored in the following manner:

An aqueous solution of p-amino-phenylalanine amide (10 mg/ml) wasadjusted to the desired pH in the range 5.5 to 9.5 with either diluteNaOH or dilute HCl as necessary, and then freeze dried. This productcould then be dissolved in water to produce a self-buffered solution atany desired concentration in the range from 0.1 M to saturation. Foreach set of conditions 4.5 μl of a solution of zinc-free insulin (20mg/ml in 0.01 N HCl) was taken. 5.5 μl of buffer (0.1 M sodiumphosphate) were added at the desired pH. The p-amino-phenylalanine amidewas then added in the form of 6.5 μl of self-buffered solution at theappropriate pH and the chosen concentration. Carboxypeptidase Y (1 μl ofan aqueous solution of 2.14 mg protein/ml) was then added. If thesolution was not clear (i.e. close to the isoelectric point of insulin),sufficient solid urea was added to clarify it. This system was used forthe rapid exploration of a range of amide concentrations and reactiontimes. For each time point the degree of coupling and degradation wasassessed in the first instance by quenching 2 μl of the reaction mixturein 100 μl of glacial acetic acid, diluting it to 1.7 ml in 0.01 N HCl,and applying 1 ml of the resulting solution for HPLC. Indications ofsuccess were confirmed by tests on the product isolated from largerscale digests after acid quenching by gel-filtration in 1% acetic acid.The reaction chosen was a Schiff base coupling with benzaldehyde andconsecutive reduction with cyanoborohydride.

The best conditions found are: pH 8.5; a final concentration ofp-amino-phenylalanine amide of 1.3 M; incubation from 7 to 22 hours at20° C. Digestion at pH values higher than pH 8.5 led to much slowerreaction, whilst digestion at pH values lower than pH 8.5 ledprogressively to more degradation and less useful synthesis. At pH 5.5,or at practically any pH in the absence of p-amino-phenylalanine amide,the concentration of carboxypeptidase Y used in the above tests led torapid and extensive degradation.

Additional experiments indicated that contrary to what is advantageouswhen using trypsin as enzyme the addition of butane-1,4-diol up to 50%by volume gives little advantage in carboxypeptidase-mediated couplingswith the alpha amino group of p-amino-phenylalanine amide, but increasesthe coupling yield when the attacking nucleophile is a benzylaminederivative.

The feasibility of the carboxypeptidase Y approach having beendemonstrated with insulin, conditions will have to be optimized for eachnew protein. With relatively few trial experiments, it will provepossible to find conditions that give useful yields of derivativescapable of coupling.

Once made in bulk, the butane-1,4-diol solutions can be stored for verylong periods. Aminobenzaldehydes being well known for the spontaneouspolymerization between their amino and aldehyde groups, had to beprotected at the aldehyde function until the amino function wasprotected by combination with the protein. The acetal protection wassufficiently stable to survive all the steps of the synthesis, but theresulting protein aldehyde acetals are so labile to acid that they canbe deprotected under conditions mild enough to present no risk to theintegrity of most proteins.

Another preferred selected region for the introduction of acomplementary group (amino or carbonyl function) is the amino terminusof the protein or polypeptide.

Thus an N-terminal glycyl residue of a protein may be converted into analdehyde function, by transamination, preferably by reaction withglyoxylate. This reaction proceeds under relatively mild conditions (seee.g. Dixon, H. B. F. and Fields, R., Methods in Enzymology, 25, 409-419[1979]). The generality of this reaction may be extended by deliberatelyintroducing Gly as N-terminal residue of proteins produced byrecombinant DNA methods. In cases where Gly is not N-terminal, a usefulketo group may nonetheless be formed by transmination of anotherN-terminal amino acid than glycine to yield the corresponding keto acid.

Furthermore, N-terminal Ser and Thr residues may be oxidized in anexceedingly mild reaction with periodate (e.g. 20° C. 26 μM protein, 1mM imidazole buffer pH 6.95, 2-fold excess of periodate for 5 min).N-terminal Ser reacts about 1000 times as fast as other protein groups(Fields, R. and Dixon. H. B. F., Biochem. J. 108, 883 [1968]), so greatspecificity may be obtained. For greater generality, N-terminal Ser orThr may be introduced by recombinant DNA techniques, or, in appropriatecases, by selecting a source of the protein of interest which has anatural Ser or Thr N-terminus.

The polypeptidyl N-terminal aliphatic aldehydes produced by thesetechniques may be reacted, preferably, with aromatic amines or withO-alkyl-hydroxylamines.

This invention also encompasses intermediate compounds of the formulaA—X—R^(1′) (II′) and R^(2′)—X′—B′ (III′), in which A, X and X′ are asdefined above, and R^(1′) and R^(2′) are defined as R¹ and R² aredefined above, except that they may additionally be protected aminogroups. B′ in formula III′ is a residue of DTPA, ferrioxamine B ordesferrioxamine B, cuprioxamine B, polyglutamic acid and derivativesthereof or [N^(ε) (DTPA-alanyl)-Lys]_(n), with n being an integer >1.

As discussed above (see page 10), preferred compounds of formula II arethose wherein X is an aromatic or araliphatic radical or has O adjacentto the amino group where R¹ is amino or protected amino. Anotherpreferred group of compounds of formula II are derivatives ofimmunoglobulins, i.e. those wherein A represents the residue of animmunoglobulin molecule, preferably of an IgG or antibody molecule, orof a fragment thereof such as an Fab or F(ab′)₂ fragment. Thepreparation of the novel compounds can be effected according to methodswell-known in the art, especially in the way described hereinbefore byreverse proteolysis.

The reaction partners of compounds II are compounds of formula R²—X′—B(III) wherein R², X′ and B are as defined above. If two proteins or aprotein and a polypeptide are to be linked together (formation of homo-and hetero-dimers) B is the residue of a protein or a polypeptide. Theproteins or polypeptides are coupled via an amino or carbonyl functionalready present in the molecule or which is introduced by methods knowin the art. A formyl function may be present in protected form as anacetal, preferably in form of a methyl or ethyl acetal.

The keto, aldehyde or acetalized aldehyde function in compounds II undIII may be introduced either directly using reactions well known in theart or indirectly in the form of a non-carbonyl precursor group whichcan be converted into a carbonyl function by known methods, such as theperiodate oxidation of a diol residue (see Examples 7 and 17) or of aresidue with vicinal hydroxy and amino groups.

Regarding the reaction partners of compounds II, i.e. the compounds offormula III as defined above, those compounds are of most importance inconnection with the present invention wherein B is the residue of achelating agent (chelon). Any compound which is capable of chelatingmetal ions can be used. If the chelating agent does already contain agroup R², i.e. an amino or carbonyl function (in which case X′ isabsent), there is no need to introduce an additional functional group ofthat type and the chelon can be coupled directly to a compound offormula II above (unless it is wished to convert an aliphatic group R²to a preferred aromatic group R²).1-p-Aminophenylethylene-dinitrilo-tetraacetic acid (U.S. Pat. No.3,994,966) is an example of such a compound which already contains anaromatic amino group. Other suitable chelating agents useful in thepresent invention and worth being mentioned are1-amino-6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosane,otherwise known as desferrioxamine or deferoxamine (in its iron-boundform known as ferrioxamine B) and diethylenetriaminepentaacetic acid(DTPA; Krejcarek et al., Biochem. Biophys. Res. Commun. 77, 581-585(1977)). These latter two chelating agents may be converted intopreferred derivatives R²—B by methods well known in the art.

Hitherto, desferrioxamine has been attached to proteins by means ofrandom coupling to side-chain amino groups, brought about either byglutaraldehyde (Janoki et al., Int. J. Appl. Radiat. Isot. 34, 871-877[1983]) or by water-soluble carbodiimide (Janoki et al., J. Nucl. Med.24, 909 [1982]). These papers, while fully demonstrating the excellenceof the choice of that chelon, also indicated the need to look formilder, more specific methods of coupling.

Ferrioxamine B is much more soluble than its iron-free form,desferrioxamine. Because it is more difficult to follow the syntheseswith the iron-free compounds, since chromatography tends to bedifficult, and there is also the danger that the hydroxylamino groups,if not protected by chelation, might participate in side reactions,ferrioxamine B instead of the iron-free compound was used to produceprotein conjugates. Then the iron was removed with acid/EDTA and themetal-free form of the conjugate was stored until needed. This approachproved satifactory, as judged by the fact that the protein conjugatetreated in this way could be loaded with ¹¹¹In and ⁶⁸Ga, whilst aprotein conjugate that still had its iron could not be loaded.Therefore, ferrioxamine B by use of the following reaction sequence(Scheme 1) was transformed to m-aminobenzoyl-ferrioxamine B which is anexample of a compound of general formula III and very useful as couplingpartner in the process of preparing protein or polypeptide derivativesin accordance with the present invention.

Instead of m-aminobenzoyl-ferrioxamine B there can also be used theanalogous compound wherein the iron ion is replaced by Cu²⁺. This latterion is sufficiently weakly bound to be replaced by other metals such asFe³⁺ but otherwise strongly enough to remain in the complex during allthe other operations described above.

The preparation of m-aminobenzoyl-ferrioxamine B and its coupling to aninsulin derivate are described in detail in Examples 1(b) and (c)respectively (below). The copper analogues of these compounds can bemade in an analogous manner to that described therein.

Cuprioxamine B is made precisely analogously to the published method forthe iron complex (Prelog, V. and Walser, A., Helv. Chim. Acta 45,631-637 [1962]) with an equivalent quantity of cupric chloride insteadof ferric chloride used in that publication. The intermediate products,as well as the final m-aminobenzoyl-cuprioxamine B are all light greenin colour. Their R_(f) values on thin-layer chromatography (t.l.c.) areall identical to those of the iron compounds.

Unlike in case of m-aminobenzoyl-ferrioxamine B, removal of the metal isnot necessary before loading with another metal in the case ofm-aminobenzoyl-cuprioxamine B. The binding constant of copper in thecomplex is many orders of magnitude lower than that for metals such asiron and gallium, which displace the copper almost instantly in dilute,neutral or mildly acid solution.

DTPA has usually been attached to proteins either by means of itsbis-anhydride (e.g. Layne et al., J. Nucl. Med. 23, 627 [1982]), or by amixed-anhydride method (Krejcarek and Tucker, Biochem. Biophys. Res.Comm. 77, 581 [1977)). In both cases, the coupling to the side chains ofthe target protein is random. In addition the bis-anhydride is capableof reacting with more than one amino group at a time, and does so to aconsiderable extent. This reagent is also very rapidly hydrolysed inaqueous media, and although this has not greatly hindered itsexploitation so far, it could nonetheless be a complicating factor undersome circumstances. Paik et al., J. Nucl. Med. 24, 932 (1983) formed amixed anhydride between carefully controlled quantities of DTPA andisobutylchloroformate. However, as with the original work of Krejcarekand Tucker (supra) they were unable to avoid the formation ofstatistical mixtures of products, no matter what ratio of reactants waschosen. The mixed anhydride, too, was labile to water.

Therefore, in accordance with the present invention an activatedderivative of DTPA was chosen that, because of its stability, could bepurified from its bi-reactive form. This derivative isDTPA-mono-(m-formylanilide) (or its dimethyl or diethylacetal) thepreparation of which is shown in the following scheme.

While mention was made above of the desirability of keeping ferrioxamineB in its metal-bound form until the end of the synthesis of theprotein-chelon conjugate, no such difficulty presents itself with theDTPA derivative. The synthesis can be carried out with metal-freecompounds, and the final protein conjugate could be loaded with thedesired ion without difficulty.

An example of very powerful compounds of formula III in terms ofchelating activity, containing a polymeric chelon, ready to couple bythe Schiff base method of the present invention are compounds of theformula m-NH₂—C₆H₄—CO—[N^(ε)-(DTPA-alanyl)-Lys]_(n), wherein n is areinteger >1. The preparation of such a compound with n=5 and its couplingis described in detail in Example 6 below. Another compound of that typeis, e.g., polyglutamic acid to which ferrioxamine B is coupled up to oneferrioxamine B per side chain carboxy group (see Example 15 below).However, in analogy to polyglutamic acid and derivatives thereof otherpolymeric compounds, especially polypeptides, may be used to formcompounds of formula III of the present invention.

Finally, another monovalent derivative of the chelating group DTPA whichcan be used for labelling of polypeptides and proteins isDTPA-alanine-p-nitrophenylester, i.e. a compound of the followingformula

This compound can be prepared in the following manner:

To 2 ml of aqueous 1 M sodium acetate buffer, pH 5.5, were added 50 mgof Ala-p-nitrophenylester.HCl under vigorous vortex mixing at roomtemperature. As soon as the material had dissolved, 84 mg ofDTPA-bisanhydride were added under further vigorous vortex mixing. Theanhydride dissolved over a period of about two minutes. At this pointthe reaction mixture was injected onto a preparative HPLC column (250×16mm, packed with 7 μm LICHROSORB RP-8 particles) previously equilibratedwith 0.1% (w/v) trifluoroacetic acid in water. The column was eluted at2 ml/min with the same solvent for 5 min, whereupon a biphasic lineargradient of pure acetonitrile was applied, the first phase reaching 35%acetonitrile after 35 minutes and the second reaching 55% acetonitrileafter 85 minutes total time. The eluent was held at 55% acetonitrile for5 minutes before being programmed down linearly to 0% over 10 min. Theeffluent was monitored at 214 nm. The desired product was collected atretention time 57-62 min and the dimeric product, due to the acylationof two molecules of Ala-p-nitrophenyl ester by the bis-anhydride, elutedat retention time 74-95 min. After removal of acetonitrile at roomtemperature on a rotary evaporator, the product was recovered bylyophilisation (yield ca. 40 mg). The product was examined by FAB-MS andby analytical HPLC on a RADIALPAK μBONDAPAK C-18 cartridge with a lineargradient of pure acetonitrile (0-60%, v/v, 2% per min.) after 5 minutesin 0.1% (v/v) aqueous CF₃COOH. The desired product eluted at retentiontime 25 min under these conditions. Some batches which contained acontaminant, identified by FAB-MS as possessing an extra alanyl residuebut only one nitrophenylester group and eluting from the analyticalcolumn at retention time 26 min were repurified on the preparativecolumn (see Example 1(c)). Yield from 50 mg Ala-p-nitrophenylester.HCl:ca. 24 mg. The final product was pure by analytical HPLC and gave theexpected FAB-MS spectrum (protonated molecular ion at m/z 586,sodium-cationised ion at m/z 608 and potassium-cationised ion at m/z624).

The above mentioned compounds, wherein a chelating agent has beenmodified in order to make it suitable for a condensation reactionyielding Schiff bases, are still novel and, therefore, are also part ofthe present invention.

EXAMPLES

The following examples illustrate but in no way limit the presentinvention.

Example 1 (a) Preparation of des-Ala^(B30)-insulin-B29-m-formylanilide

Tris-HCl (16 mg) was crushed in 3 ml butane-1,4-diol contained in acentrifuge tube. The suspension was thoroughly mixed and the crystalsthat had not dissolved were centrifuged to the bottom of the tube. To1.162 ml of the supernatant was added 260 mg of m-aminobenzaldehydedimethyl acetal, and the mixture was agitated on a Vortex mixer. (In asecond experiment the corresponding diethyl acetal was used.) Afteraddition of 30 μl of N-ethylmorpholine (redist.) and further agitation,the pH of the solution was measured with a glass electrode and adjustedto 6.2 by successive additions of 10% (v/v) acetic acid inbutane-1,4-diol. Because of the acid-lability of the acetal, the acidwas added very cautiously, with vigorous mixing. Particular care wasneeded once the pH had fallen below 6.5. The resulting solution could bestored frozen, at −20° C., for many months.

The buffered acetal solution (300 μl) was added to 20 mg of Zn-freeinsulin (Rose et al., Biochem. J. 211, 671 [1983]), followed by 10 μl ofwater. The insulin normally went into solution during a 1 h incubationat 38° C., with only occasional, mild agitation. Once the insulin was insolution, 10 μl of a freshly made solution of TPCK-treated trypsin (5 mgin 40 μl) was added, and incubation was continued at 37° C. The progressof the coupling was followed by cellulose-acetate electrophoresis (pH 8)as described by Rose et al., Biochem. J. 195, 765 [1981] or by HPLC(250×4.6 mm RP-18 SPHERI 5 column with a linear gradient of 3.5 to 35%(v/v) acetonitrile in 0.3 M ammonium sulfate in 7 minutes). The reactionproduct migrated more slowly than insulin on electrophoresis, andemerged two minutes after insulin on HPLC. As judged by eithercriterion, the ratio of insulin to product was about 1:2.5 in favour ofthe latter after, typically, 3 h of incubation. As seen by HPLC someother protein peaks appeared progressively during the incubation, butthe largest of them only represented 14% of the total protein by theend.

The reaction mixture was cooled and 3.1 ml of glacial acetic acid wereadded to stop the enzymic reaction. The resulting mixture was thendiluted with an equal volume of 10% (v/v) acetic acid and subjected togel filtration on a 90×2.6 cm column of SEPHADEX G-50 (fine), elutionwith 1% acetic acid.

The fractions containing the insulin derivative were pooled andlyophilized. The product was further purified by ion-exchangechromatography (column 2×20 cm A-25, Pharmacia) equilibrated with 7Murea-100 mM tris, adjusted to pH 8.4 (glass electrode, urea alreadypresent) with 12M HCl. Elution was carried out by means of a lineargradient (1 liter total) between this starting buffer and one identicalexcept for the presence of NaCl (200 mM). The fractions is of the firstpeak to emerge were pooled and dialysed against 1% (v/v) acetic acid.

If it was wished to preserve the acetal (normally only in the case ofthe slightly more stable methyl form), then the trypsin was stopped bydiluting the incubation mixture into 5 ml of the A-25 column startingbuffer and applying the solution directly to the A-25 column. In thiscase most conditions were as given above except that the gradient wasfrom 0 to 155 mM NaCl. The trypsin passed through the column at once,together with the m-aminobenzaldehyde dimethylacetal and its sideproducts. The wanted product emerged early in the gradient, about 4column volumes after the initial breakthrough. Assay showed that thedesired product was nonetheless uncontaminated by any trypsin activity.With the exception of unchanged insulin, which emerged much later in thegradient, the wanted product was the only significant protein peak. Thepooled product fractions were dialysed against 0.5% (w/v) aqueousNH₄HCO₃ and freeze-dried.

The products could be further purified (from rather minor contaminants)by HPLC (250×4.6 mm RP-18 Spheri 5 column with a linear gradient of 3.5to 35% (v/v) acetonitrile in 0.3 M ammonium sulfate in 7 minutes),loading of up to 5 mg at a time. The eluant was sufficiently acidic todeprotect any acetalized protein during the separation. Thechromatographic behavior of acetalized and non-acetalized insulinderivatives was identical.

Characterization of the product:

The compound obtained (after acid-treatment to remove putative acetalprotection) was coupled to m-aminobenzoic acid to yield a Schiff base inthe usual way (see Example 1(c)). If tritiated borohydride was used toreduce the Schiff base, tritium was incorporated into the proteinfraction. The reduced product migrated on cellulose-acetate (pH 8) asthough it had regained the —COOH group lost by replacement of theC-terminal alanine by the m-formyl-benzaldehyde, and the band of reducedproduct showed the characteristic blue fluorescence of m-aminobenzoates.Digestion with Armillaria protease gave a product with the expectedpaper-electrophoretic properties.

(b) Preparation of m-aminobenzoyl-ferrioxamine B

Ethyl acetate (10 ml) was added to 125 mg of m-aminobenzoic acid(purum). N-Ethyl morpholine (redist., 200 μl) was added to the mixture,followed by 1 ml of di-tert.-butyl dicarbonate (purum). The faintlycloudy solution clarified on stirring overnight at 20° C. The mixturewas then extracted three times with 10 ml of 0.3 M NaHCO₃ and the pooledaqueous layers were cooled and acidified by addition of an equal volumeof cooled 0.6 M citric acid. A precipitate formed at this point, whichdisappeared when the suspension was extracted with 15 ml of ethylacetate. After separation of the phases, the aqueous layer wasre-extracted with 5 ml of ethyl acetate and the two organic layerscombined. The pooled organic fraction was allowed to stand overnightover MgSO₄. The solution was then dried, first by rotary evaporation,then in a desiccator (NaOH pellets) under an oil-pump vacuum. Some 80 mgof this product were dissolved in 252 82 l of dimethylformamide,together with 42.6 mg of N-hydroxysuccinimide. To this solution wereadded 76.4 mg of dicyclohexylcarbodiimide in 63 μl of dimethylformamide,and the mixture was allowed to stand overnight under agitation. Aprecipitate began to form at once, and by the following morning wasextremely dense. TLC of the supernatant showed virtually completeconversion to a product having the expected migration of aBoc-m-aminobenzoic acid hydroxysuccinimido ester. The precipitate wasremoved by centrifugation and the supernatant dried in a vacuumdesiccator over NaOH pellets under an oil-pump vacuum. The slightly waxysolid thus obtained was used in the following reaction without furtherpurification.

To 4 ml of a 0.4 M aqueous solution of ferrioxamine (343.8 mg) was addedan equal volume of dimethylformamide. N-Ethylmorpholine was then addeduntil the pH (estimated externally on moist pH paper) rose above 8.About 300 μl of the base were required. To this mixture was then added196 mg Boc-m-aminobenzoic acid hydroxysuccinimido ester dissolved in 4ml of dimethylformamide. After ascertaining that the pH had not changednoticeably, the mixture was left overnight at 20° C. The followingmorning 12 ml of distilled water were added to the reaction mixture, andthe solution was extracted three times with 24 ml (each time) ofchloroform. The pooled organic layers were extracted twice with 3.2 ml(each time) of water. The organic phase was dried on a rotaryevaporator. The product was characterized by FAB-MS. The Boc-group wasremoved by dissolving the residue in 7 ml of 98-100% formic acid. After30 min. the deprotected product was isolated by HPLC (250×16 mm columnmfilled with 7μ LICHROSORB RP-8, linear gradient between 15% and 45%acetonitrile over 30 min. in 0.1% (v/v) aqueous CF₃COOH, at a flow rateof 2 ml/min). The desired product (222 mg, 54% yield) was in the firstmajor peak (retention time 35.5 minutes) to emerge after the injectiontransient and was characterized by FAB-MS. A second peak emerged after42.5 minutes, and was found by mass spectrometry to be the N-formylderivative of the desired product. A third peak (retention time 56.5min) contained non-deprotected starting product.

The degree of N-formylation can be considerably reduced by working at alower concentration of the Boc-derivative (12 mg/ml). It is alsopossible to remove the formyl group by treatment with 0.1 M HCl at roomtemperature, monitoring the progress of the removal by HPLC.

The above preparation can be followed by TLC. In n-butanol/aceticacid/pyridine/water (15:3:12:20, v/v) on KIESELGEL 60 (Merck) therespective R_(f)-values are:

ferrioxamine 0.27 m-aminobenzoyl-ferrioxamine B 0.44N-formyl-m-aminobenzoyl-ferrioxamine 0.48Boc-m-aminobenzoyl-ferrioxamine 0.56

(c) Preparation of des-Ala^(B30)-insulin/ferrioxamine B Conjugate

1 mg of des-Ala^(B30)-insulin-B29-m-formylanilide was dissolved in 45 μlof a 5.5 mM solution of m-aminobenzoyl-ferrioxamine B in a 1% (v/v)aqueous acetic acid buffer brought to pH 3.5 with strong NaOH solution.To this solution was added 20 μl of aqueous 2.4 mM sodiumcyanoborohydride and 50 μl of water.

After 20 min. it was checked by HPLC that the reaction was terminatedand the reaction product,des-Ala^(B30)-insulin-B29-NH—C₆H₄-m-CH₂—NH-m-C₆H₄—CO-ferrioxamine B, wasisolated by HPLC using a 250×4.6 mm RP-18 SPHERI 5 column (BrownleeLaboratories, 2045 Martin Ave., Santa Clara, Calif. 95050, USA) and alinear acetonitrile gradient in 0.3 M aqueous ammonium sulfate over 30min. The gradient was made by a two pump system with 0.3 M ammoniumsulfate as solvent A (equilibration) and 0.3 M ammonium sulfate/35%(w/v) aqueous acetonitrile. The two solvents were made up using stocksolutions of 3 M ammonium sulfate which had been adjusted to anindicated pH of 2.7 (glass electrode) by careful addition of conc.H₂SO₄. Elution was at 1 ml/min.

After removal of acetonitrile in a current of air, the is pooledfractions containing the insulin conjugate (a pale red solution) wereworked up on a SEP PAK cartridge (Waters Associates, Milford, Mass.01757, USA) according to the manufacturer's instructions. After a firstwash with 10% (v/v) aqueous acetonitrile elution was effected with 40%(v/v) aqueous acetonitrile. The concentrated product was lyophilizedafter blowing off the acetonitrile. The peach-coloured solid was foundto be homogeneous on cellulose acetate (pH 8) and HPLC RADIALPAK μBONDAPAK C-18 cartridge in a Z-module, linear gradient of pureacetonitrile (0-60%) in 0.1% (v/v) aqueous CF₃COOH). The product wasfurther characterized by FAB-MS of Armillaria protease digest. Digestionwas carried out at pH 7.8 in 1% (w/v) ammonium hydrogen carbonate. Thisprotease is known to cleave specifically on the amino-terminal side ofLys^(B29) of insulin.

Example 2 Removal of Iron from the des-Ala^(B30)-insulin/ferrioxamine BConjuate

To 225 μl of a 138 μM solution in water of thedes-Ala^(B30)-insulin/ferrioxamine B conjugate obtained according toExample 1 were added 25 μl of 1 M propionic acid and 2 μl of 0.01 M EDTAand the mixture was adjusted to pH 3 with diluted HCl. The pink colourof the solution was rapidly lost. If desired the reaction can befollowed spectroscopically, since after removal of iron the peak between400 nm and 450 nm, characteristic of the ferrioxamine B conjugate willdisappear completely. Such spectroscopy with a test solution of 1 mMferrioxamine B showed that the half-time for the removal of iron at pH 3was approximately 90 seconds, whilst that at pH 3.5 was approximately340 sec. The reaction at pH 2.5 was too rapid to follow.

Once the iron had been removed, the solution was adjusted to pH 5.5 with1.7 M sodium acetate buffer. The resulting precipitate was carefullywashed by centrifugation in 0.7 M sodium acetate buffer, until theresidual EDTA concentration could be expected to be negligible comparedto that of the conjugate. It was found that such traces as persistedcould not compete significantly for ⁶⁸Ga and ¹¹¹In under the labellingconditions described below. The iron-free protein derivative was storedas a frozen pellet for months without apparent impairment of itscapacity to be labelled.

Example 3 Labelling of the des-Ala^(B30)-insulin/desferrioxamine BConjugate with Radioactive Metals

The des-Ala^(B30)-insulin/desferrioxamine B conjugate obtained accordingto Example 2 was dissolved or suspended at a concentration of 7 μg/μl ina buffer made by mixing equal volumes of 0.1 M ammonium acetate and 0.1M sodium citrate and adjusting the resulting solution to pH 8.5-9 with33% (w/v) aqueous ammonia. The radioactive metal solution (¹¹¹In Cl₃ or⁶⁸GaCl₃) was added to the buffer solution at a concentration of between1 μCi and 1 mCi/μl just before the protein. The reaction mixture can beused directly in all further tests.

The degree of labelling can be determined immediately using radioactivetechniques such as scintillation radioactive counting, radioactivityscanning, and radioautography on standard X-ray film after HPLC orcellulose acetate electrophoresis (pH 8).

Example 4 (a) Preparation ofdes-Ala^(B30)-insulin-B29-p-aminophenylalanine-methylester

p-Amino-L-phenylalanine-hydrochloride.1/2 H₂O (Bachem, Bubendorf,Switzerland) was dissolved with agitation in 8.5 M HCl/MeOH at about 140mg/ml and incubated overnight at room temperature. The reagents wereremoved under a stream of nitrogen and finally under high vacuum toyield as a crisp white powderp-amino-L-phenylalanine-methylester-hydrochloride. The reaction wasquantitative as determined by TLC (butanol/acetone/acetic acid/water,7:2:7:4, v/v) and cadmium-ninhydrin as the stain.

The R_(f) of the starting material was 0.28; that of the product was0.52.

208 mg of p-amino-L-phenylalanine-methylester.HCl were dissolved in 2 mlof butane-1,4-diol. To this solution were added approximately 2 ml of0.5 M tris-base in butane-1,4-diol/H₂O (4:1, v/v), until the pH (glasselectrode) was 6.5. 100 mg of solid Zn-free insulin were added to 3.1 mlof the buffered methyl ester solution. Most of the insulin went intosolution after incubation at 37° C. for 30 minutes. Trypsin (12 mg,Worthington TPCK grade) was dissolved in 120 μl of water and 100 μl ofthe solution were added. After 90 minutes at 37° C., during which timethe insulin dissolved completely, cellulose-acetate electrophoresis atpH 8 revealed that more than half of the insulin had been converted to aproduct that migrated more slowly than insulin. Since the pK of anaromatic amino group is well below 8, this is the expected behaviour ofthe desired product. The reaction mixture was cooled and 3.1 ml ofglacial acetic acid were added to stop the enzymic reaction. Theresulting mixture was then diluted with an equal volume of 10% (v/v)acetic acid and subjected to gel filtration on a 90×2.6 cm column ofSephadex G-50 (fine), elution with 1% (v/v) acetic acid.

The fractions belonging to the peak of the derivative were pooled andlyophilized. The product was further purified by ion-exchangechromatography (column 2×20 cm A-25, Pharmacia) equilibrated with 7 Murea-100 mM tris, adjusted to pH 8.4 (glass electrode, urea alreadypresent) with 12 M HCl. Elution was carried out by means of a lineargradient (1 liter total) between this starting buffer and one identicalexcept for the presence of NaCl (200 mM). The first peak to emerge waspooled and dialysed against 1% (v/v) acetic acid. After lyophilization,54.5 mg of des-Ala^(B30)-insulin-B29-p-aminophenylalanine-methylesterwere obtained.

The product gave a single peak on HPLC RADIAPAK μ BONDAPAK C-18cartridge in a Z-module, linear gradient of 25-45% (v/v) acetonitrile in0.1% (v/v) aqueous CF₃COOH over 20 minutes). The product migrated oncellulose-acetate electrophoresis at pH 8 as a single spot, in theexpected position, i.e. migrated more slowly than insulin. Onelectrophoresis at pH 1.9, well below the pK of an aromatic amino group,the product migrated faster than unmodified insulin, consistent with itspossessing an extra positive charge at this pH. Digestion of the productwith trypsin released des-octapeptide-(B23-B30)-insulin (which wasidentified by HPLC and by electrophoresis on cellulose-acetate at pH 8),the heptapeptide comprising residues B23-B29 (which was identified byHPLC, by paper electrophoresis at pH 6.5, and by FAB-MS) and freem-aminophenylalanine-methyl-ester (which was identified by HPLC and bypaper electrophoresis at pH 6.5).

(b) Preparation of DTPA-mono-(m-formylanilide)

Both Dowex-50 (WX-4, H-form) and pumice boiling stones were washed byfiltration in 20 volumes of ethanol. Both were then boiled in 5 volumesof ethanol for 3 min., filtered, and dried in a vacuum desiccator. To 1g of dried DOWEX-50 was added 13.5 g m-nitrobenzaldehyde (purum) and acopious quantity of boiling stones. Methanol (125 g) was added and themixture was refluxed for 60 min. The mixture was then cooled andfiltered. Approximately 70 ml of 0.2 M sodium carbonate-bicarbonatebuffer, pH 9.5, were added. The aqueous phase of the resulting emulsionwas extracted with 40 ml and 35 ml of ethyl acetate. The organic layerwas dried for some hours over freshly baked K₂CO₃, filtered andevaporated to a syrup. TLC on KIESELGEL 60 (Merck) with CHCl₃/MeOH (9:1,v/v) showed a change in R_(f) consistent with complete conversion to thedimethyl acetal (R_(f)=0.68 before, 0.73 after).

In an analogous way by refluxing 13.5 g of m-nitrobenzaldehyde with 125g of ethanol for 30 min. the corresponding diethyl acetal was preparedwhich was easier to isolate. Since ethanol forms azeotropic mixtureswith water, 60 ml could be removed by distillation while the DOWEXcatalyst was still present (there was no need to add alkali). Theresidue, after filtration, was taken down to a syrup on a rotaryevaporator.

The reduction of the nitro to the amino compound followed the methodproposed by Howarth and Lapworth, J. Chem. Soc. 121, 76-85 (1922), forthe diethyl acetal.

50 g of Na₂S (puriss.) were dissolved in 50 ml of water and 25 g of 12 MHCl were added slowly, with stirring. The resulting solution was added,with stirring, to a solution of 15 g of the dimethyl acetal in 90 ml ofmethanol (ethanol for the diethylacetal). Reaction was completed byrefluxing for 6 h, after which the alcohol was distilled off from thedeep red solution. The aqueous residue was cooled and extracted twicewith 30 ml of diethylether. The ether layer was dried over MgSO₄,filtered and dried by rotary evaporation. The dimethyl and diethylacetals could be stored, over periods of several months, without visibledecomposition or polymerization, at room temperature in the dark.

To 20 mg of m-aminobenzaldehyde diethyl acetal was added 1 ml ofpyridine followed by 1 ml of water. The resulting solution was slowlyadded to 200 mg of diethylene-triamine pentaacetic acid (DTPA)dianhydride (Calbiochem, La Jolla, USA) whilst Vortex mixingcontinuously to dissolve the anhydride.

After standing 15 min. at room temperature, the solution was cooledexternally with ice whilst 2 ml of acetic acid were added. After 1 h atroom temperature the sample was applied (two separate runs) to an HPLCsystem which has already been described by Rose et al., Biochem. J. 220,189 (1984). A RADIALPAK μBONDAPAK C-18 cartridge was used, equilibratedwith 0.1% (v/v) aqueous trifluoroacetic acid. The flow rate was 2ml/min. Once the pyridine acetate had eluted (monitoring absorbance at214 nm) after 30 min., a linear gradient of pure acetonitrile (increase2% per min. up to 60%, v/v) was applied to elute the two majorfractions. Together these fractions account for more than 90% of theabsorbance at 214 nm appearing after the reagent front, and they are theonly ones to give a precipitate with 2,4-dinitrophenyl hydrazine. Rotaryevaporation of the acetonitrile followed by lyophilisation yielded 9 mgof first fraction and 8.5 mg of second fraction. Analytical high voltagepaper electrophoresis was performed at pH 6.5. The spots, ninhydrinnegative, were revealed by spraying with a saturated solution of2,4-dinitrophenylhydrazine in 2 M HCl.

Paper electrophoresis at pH 6.5 showed the first HPLC fraction to have arelative mobility (m) of −0.72 (m of aspartic acid=−1.0), and the secondfraction had an m-value of −0.34. The first HPLC fraction was consideredto be the result of acylation of one molecule of m-aminobenzaldehydefollowed by hydrolysis of the second anhydride moiety, and the secondHPLC fraction was considered to be the result of acylation of twomolecules of m-aminobenzaldehyde by the DTPA-dianhydride. Thisinterpretation was confirmed by the FAB-MS spectra of the two fractions,which contained signals due to protonated molecular ions at m/z 497 and600, respectively, and showed that, as expected, the acetal protectionhad been removed by the acidic conditions of the work-up.

(c) Coupling Betweendes-Ala^(B30)-insulin-B29-p-aminophenylalanine-methylester andDTPA-mono-(m-formylanilide)

The DTPA-m-aminobenzaldehyde was dissolved in the pH 3.5 buffer ofExample 1(c) at a concentration of 5 mM (2.64 mg/ml). 10 mg ofdes-Ala^(B30)]insulin-B29-p-aminophenylalanine-methylester weredissolved in 450 μl of this solution and 500 μl of 10 mM NaBH₃CN wereadded. A precipitate (later identified as the desired product) began toform at once. After 15 minutes the precipitate was brought back intosolution by cautious addition of glacial acetic acid, and diluted toapproximately 1.5 ml with 0.1% (v/v) aqueous CF₃COOH. The mixture wassubjected to HPLC RADIALPAK μBONDAPAK C-18 cartridge in Z-module,isocratic 5 min., then linear gradient of 25-45%, v/v, acetonitrile in0.1%, w/v, aqueous CF₃COOH over 20 min.). The desired product emergedafter about 17 minutes, and was the first peak after the injectiontransient. The acetonitrile was evaporated in a stream of nitrogen and,after lyophilisation, 9 mg of product were obtained. The product washomogeneous on HPLC and also on cellulose-acetate electrophoresis at pH8. It gave a spot in the latter system which migrated faster thaninsulin towards the anode. After labelling with excess non radioactiveIn^(III) and work-up on a SEP PAK, the product was characterized bydigestion with trypsin followed by HPLC.

Example 5 Labelling of thedes-Ala^(B30)-insulin-B29-p-aminophenylalanine-methylester/DTPA-mono-(m-formylanilide)Conjugate with Radioactive Metals

Thedes-Ala^(B30)-insulin-B29-p-aminophenylalanine-methylester/DTPA-mono-(m-formylanilide)conjugate obtained according to Example 4 was labelled with ¹¹¹In and⁶⁸Ga in the same manner as described in Example 3 for thedes-Ala^(B30)-insulin/deferoxamine conjugate.

Example 6 (a) Preparation of m-aminobenzoylpenta-[(N^(ε)-DTPA-alanyl)-lysine]

N^(α)-Carbobenzoxy-penta-[(N^(ε)-Boc)-lysine] (Bachem, Switzerland) wasdissolved in anhydrous trifluoroacetic acid at a concentration of 25 mgin 750 μl. After 30 min. at room temperature, the trifluoroacetic acidwas removed by evaporation. Further traces of acid were removed byredissolving the solid in anhydrous methanol (10 μl of MeOH for every mgof starting product). The product now gave a ninhydrin-positive spot onpaper electrophoresis, having the expected mobilities at pH 1.9 and 6.5.A quantity of the dried acid-treated material equivalent to 8.5 mg ofstarting product was dissolved in DMSO (21 μl), 34 mg ofDTPA-alanine-p-nitrophenyl ester were added, followed by 42 μl of DMSO.The apparent pH was then adjusted to 8 (moist pH paper) with N-ethylmorpholine (approx. 35 μl).

The reaction was followed by paper electrophoresis at pH 6.5 and wasjudged complete after 25 h (progressive replacement of the startingproduct with ever more acidic spots with ever fainter ninhydrin colour,finally virtual disappearance of all ninhydrin colour). The reactionmixture was lyophilized and re-dissolved in conc. aqueous HBr (45 μl foreach mg of the protected penta-lysine starting material). After 30 min.at 20° C., the sample was dried down. It was then re-dissolved in 1%acetic acid (at a concentration of 10 mg/ml) and passed down a column ofSEPHADEX G-25 (8 mm internal diameter, 60 cm long). The wanted product,penta-[(N^(ε)-DTPA-alanyl)-lysine], emerges in the breakthrough volume.It had the expected mobility on paper electrophoresis at pH 6.5. TheSEPHADEX pool was lyophilised A quantity of this product, equivalent to10 mg of protected penta-lysine, was taken up in 166 μl DMSO and 26 mgof Boc-m-aminobenzoyl-hydroxysuccinimido-ester added, followed by 4 μlN-ethyl-morpholine. After 15 h at 20° C., the original ninhydrin colouron electrophoresis disappeared. After treatment of the dried reactionmixture with 750 μl trifluoroacetic acid and subsequent drying, theninhydrin colour returned at approximately the same electrophoreticposition (as expected) but as a much fainter, but much more rapidlydeveloping, yellow colour (characteristic of an aromatic as opposed toan aliphatic amino group).

(b) Preparation of Conjugate between m-aminobenzoylpenta-[(N^(ε)-DTPA-alanyl)-lysine] and des-Ala^(B30)-insulin

The crude product obtained under (a) was used directly as a 7 mMsolution for coupling to the product of Example 1(a) in exact analogy tothe method of Example 1(c) to yield the desired conjugate. The newprotein derivative has the expected intense blue fluorescence, and amobility on cellulose-acetate electrophoresis at pH 8.3 of approximately1.5 times that of the starting insulin derivative. The product waslabelled with ¹¹¹In according to the method of Example 3. Titration ofthe product with ¹¹¹In of low specific activity using cellulose acetateelectrophoresis to distinguish between protein-bound and protein-freeindium suggested that nearly all of the DTPA groups are labelled whenthe appropriate quantity of ¹¹¹In is presented to the protein at aconcentration of approximately 10 pM.

Example 7 (a) Preparation of S-(2,3-dihydroxypropyl)-cysteamine

Cysteamine hydrochloride (1.08 g) and dithiothreitol (1.46 g) weredissolved in 100 ml of ammonium bicarbonate (2%, w/v) and allowed tostand at room temperature for 15 minutes. 3.1 g of3-bromo-1,2-dihydroxy-propane were added. The alkylation of the thiolgroup was followed by paper electrophoresis at pH 1.9 (methods describedby Gonzales and Offord, Biochem. J. 125, 309-317 (1971)). After 29 hoursat room temperature, the cysteamine spot (staining yellow withninhydrin) had been converted almost exclusively into a spot (staininggrey with ninhydrin) with the predicted mobility (Offord, MethodsEnzym., 47, 51-69 [1977]) of the wanted product. A faint second spot wasvisible that corresponded to the predicted mobility of the bis-alkylatedcysteamine.

The reaction mixture was applied directly to a column of DOWEX 50X8(3×15 cm) previously equilibrated with pyridine-acetic acid-water,25:1:225, v/v, pH 6.5). The column was then washed with 200 ml of H₂Ofollowed by 50 ml of the pH 6.5 buffer. The wanted product was liberatedfrom the column with 100 ml of ammonia solution (4M). The ammonia eluatewas rotary evaporated for 20 min. and then freeze-dried; yield 493 mg ofelectrophoretically homogeneous material.

(b) Preparation ofdes-Ala^(B30)-insulin-B29-S-(2.3-dihydroxylpropyl)-cysteamide

A sample of the product obtained under (a) (200 mg) was dissolved in1.118 ml of butane-1,4-diol. Glacial acetic acid (37.5 μl) was added.The pH (glass electrode, very slow response) was raised to 7.0 withapprox. 100 μl of a saturated solution of Tris(base) in butane-1,4-diol.

This solution was used to prepare the wanted product in a mannerprecisely analogous to that described in Example 4 (including thework-up). The protein product had the electrophoretic and ion-exchangeproperties expected of a mono-amide-substituted insulin, but itsprincipal characterization lay in its use for the Schiffbase-mediatedcoupling to m-aminobenzoic acid and the detailed study of this latterproduct (see below).

(c) Coupling ofdes-Ala^(B30)-insulin-B29-S-(2,3-dihydroxylpropyl)-cysteamide tom-aminobenzoic Acid

2 mg of the substituted insulin amide obtained according to (b) above,was dissolved in 150 μl of sodium acetate buffer (0.48 M, pH 5.6, 7 M inurea). To this were added 16 μl of freshly made up periodic acid (4 g/lH₂O) and 83 μl of a solution of m-aminobenzoic acid (1.23 M). Thislatter solution was made by adjusting a 0.1 M solution of the acid to pH6.5 with strong sodium hydroxide solution, lyophilizing andre-dissolving to 1.23 M. Finally, 16.6 μl of sodium cyanoborohydride (30mM) was added. After 60 min. the mixture was dialysed and re-dissolvedin 1 ml of HCl (0.01 M). This solution was applied for HPLC on a C18column as described in the preceding Examples and developed with alinear gradient of 29-35% (v/v) acetonitrile in 0.3 M ammonium sulphate,lasting 15 minutes. The stock ammonium sulphate solution (3 M) used tomake these eluants had been adjusted to pH 2.7 (glass electrode) withstrong H₂SO₄. The product peak, corresponding to about 90% of theprotein, emerged approx. 2 min. after the position of the startingmaterial. The protein was absorbed from the appropriate pooled fractionsonto a SEP PAK cartridge, after blowing off the acetonitrile. Afterwashing the cartridge with 0.1% (v/v) aqueous trifluoroacetic acid/10%(v/v) aqueous acetonitrile, the product was eluted with 2 ml of 0.1%aqueous trifluoroacetic acid/40% aqueous acetonitrile. The protein,recovered by blowing off the acetonitrile and then lyophilizing, wasstrongly fluorescent. It was characterized by its mobility oncellulose-acetate electrophoresis and by digestion with Armillariaprotease. The small fragment from the Armillaria digest wascharacterised by FAB-MS.

Example 8 General Procedure for the Preparation of IqGs Labelled at theC Termini of Their Chains with DTPA (a) Selection of Optimal Conditions

A monoclonal or polyclonal IgG (9 mg/ml, isotonic saline) is mixed withbuffer (Na-phosphate, 0.1 M, pH 8.5), p-aminophenyl-alanine-amide (460mg/ml), and carboxypeptidase Y (2 mg/ml) in the ratio 4.5:5.5:6.5:1 byvolume. The mixture is allowed to stand at room temperature. At chosentimes after the start of the reaction (normally after 20 min., 60 min.,2.5 h, 5 h, 18 h) samples of 6.5 μl are taken and precipitated with 2 μlof aqueous trichloroacetic acid, 10% (w/v). The precipitate is collectedby centrifugation and washed by centrifugation in a further 200 μl ofthe trichloroacetic acid solution. The pellet is dissolved in saturatedaqueous urea, precipitated and washed once more using 10%trichloroacetic acid. (This extensive washing is necessary to eliminatetraces of the p-aminophenylalanine amide, since this compound is presentat the start at a very high concentration). The pellet is dissolved inacetate buffer (acetic acid, 10%, brought to pH 3.5 with concentratedNaOH) using urea if necessary. It is then treated with 0.5 mg of solidDTPA bis-anhydride under strong agitation. After 2 min., the protein isprecipitated and washed again as above. A further cycle of solution,re-precipitation and washing is carried out. The product is thenresuspended in 10 μl of the citrate buffer of Example 3 and 1 μl of asolution of ¹¹¹In-oxime (Amersham International p.l.c., GB) is added.After 5 minutes, the pellet is collected by centrifugation, washed twicein 200 μl distilled water and counted.

Control samples are prepared in parallel at each time point. These canbe obtained from an incubation in which the carboxypeptidase Y solutionis replaced by an equivalent volume of distilled water. The optimum timeis considered to be when (after correction for the specificradioactivity of the ¹¹¹In and subtraction of the backgroundradioactivity indicated by the corresponding control) the radioactivityof the sample corresponds to each IgG molecule having an average of onep-aminophenylalanine-amide-residue at its carboxyl terminus. Arepresentative result, at the specific radioactivity of ¹¹¹In used (85μCi/μmole), is approx. 6000 cpm after subtraction of the control.

(b) Larger-scale Preparation

The x hours incubation, which gave the optimun result in part (a) isthen repeated on 0.4 mg IgG. The reaction is stopped by 25-fold dilutionto pH 3.5 (10% acetic acid previously adjusted with 5 M aqueous NaOH).The protein is recovered from this mixture by gel filtration (SEPHADEXG-150).

The resulting product is reacted with DTPA-mono-(m-formylanilide)(Example 4) with reduction by cyanoborohydride in the usual way: Proteinconcentration 4 mg/ml, aldehyde concentration 1 mM, cyanoborohydrideconcentration 0.75 mM, pH 3.5 (acetate, 1%). After 90 minutes a sampleis taken, and labelled by incubation with ¹¹¹In as described in, e.g.,Example 3. The labelled protein is separated from labelled, un-coupledaldehyde on a SEP PAK cartridge. The aldehyde is removed by 20% aqueousacetonitrile/0.1% aqueous trifluoroacetic acid whilst the protein isnot. The radioactivity associated with the protein (after subtraction ofappropriate control counts) corresponds to complete reaction. Thedesired protein is isolated from the remainder of the Schiff-basereaction mixture by gel-filtration, as described before.

Example 9 (a) Preparation ofdes-Ala^(B30)-insulin-B29-N-(formylmethyl)-amid

To 1.33 g of aminoacetaldehyde-diethylacetal (Fluka, Switzerland), wereadded 8 ml of butane-1,4-diol followed by 450 μl of acetic acid. To 100mg of des-Ala^(B30)-insulin (prepared essentially according to Moriharaet al., Biochem. Biophys. Res. Commun. 92, 396 [1980]) were added 3 mlof the acetal solution followed by 0.1 ml of water and the mixture wasincubated at 37° C. for 10 min., whereupon the des-Ala^(B30)-insulindissolved. After addition of 100 μl of water containing 10 mg ofTPCK-treated trypsin (Worthinton), the solution was incubated at 37° C.for 90 minutes and quenched at 0° C. with an equal volume of pure aceticacid. The resulting solution was diluted with an equal volume of 1%acetic acid then gel filtered on a column (90×2.6 cm) of SEPHADEX G50(fine grade) eluted with 1% (v/v) acetic acid. The fractions containingthe insulin derivative were pooled and lyophilized. The product wasfurther purified by ion-exchange chromatography on an A-25 column asdescribed in Example 4(a). After dialysis against 1% acetic acid andlyophilization, 33 mg product were recovered.

Upon HPLC, the product eluted 1.5 min. later than thedes-Ala^(B30)-insulin from the C-18 cartridge (conditions as in Example4(a)), consistent with its being more hydrophobic. Deprotection of theacetal was achieved most conveniently by incubatingdes-Ala^(B30)-insulin-B29-N-(formylmethyl)-amid-diethylacetal (2 mg/ml)in 5% formic acid at 37° C. overnight, under which conditionsapproximately 90% deprotection was achieved. Upon analytical HPLC, theproduct, des-Ala^(B30)-insulin-B29-N-(formylethyl)-amide, eluted fromthe C-18 cartridge earlier than the diethylacetal (and very close to theposition of des-Ala^(B30)-insulin). Thedes-Ala^(B30)-insulin-B29-N-(formylmethyl)-amid was recovered from thedilute formic acid by lyophilization.

(b) Preparation ofdes-Ala^(B30)-insulin-B29-N-(formylmethyl)-amide/m-aminobenzoyl-ferrioxamineB Conjugate

To 20 mg des-Ala^(B30)-insulin-B29-N-(formylmethyl)-amide dissolved in0.2 ml of 0.48 M aqueous sodium acetate buffer (pH 5.6) was added 117 mgof m-aminobenzoyl-ferrioxamine B dissolved in 800 μl ofdimethylformamide. The pH was adjusted to 5.0 with NaOH and then 500 μlof a 10 mM solution in water of sodium cyanoborohydride (Aldrich) wereadded. The pH, which had risen to 6.2, was adjusted to 5.5 and thesolution was incubated overnight at room temperature. Analytical HPLC onthe C-18 cartridge (conditions as in Example 4(a)) showed that about 80%of the starting material had been transformed to a species eluting 3minutes later than the remaining starting material. This later-elutingspecies, later identified as the desired product,des-Ala^(B30)-insulin-B29-NH—CH₂—CH₂—NH—C₆H₄-m-CO-ferrioxamine B, wasisolated preparatively on the same column. The product (11.9 mg) waspeach-coloured due to the iron present, and was characterized bydigestion with trypsin, which yielded des-octapeptide-(B23-B30)-insulin(identified by HPLC and electrophoresis on cellulose acetate at pH 8),the heptapeptide comprising residues B23-B29 (identified by HPLC, byelectrophoresis on paper at pH 6.5 and by FAB-MS), andNH₂—CH₂—CH₂—NH—C₆H₄-m-CO-ferrioxamine B (identified by FAB-MS).

Example 10 (a) Preparation of DTPA-mono-(N-formylmethyl)-amideDiethylacetal

This compound was prepared from DTPA-bisanhydride andaminoacetaldehyde-diethylacetal analogously to the preparation ofDTPA-mono-(m-formylanilide) (Example 4(b)), using 20 mg of the aminocomponent and 200 mg of the anhydride. Again, two major products wereisolated by HPLC (as in Example 4(b)), 5 mg of a first fraction and 6.1mg of a second fraction. The first fraction, upon electrophoresis onpaper at pH 6.5, had a relative mobility of about −0.76 (Asp=−1.0); itwas negative to ninhydrin and was revealed by spraying withDNP-hydrazine in 2 M HCl. Heating to 100° C. was necessary to reveal ayellow spot, suggesting that the acetal protection, much less labile foraliphatic aldehydes than for aromatic ones, was still present. Thefraction was characterized as DTPA-mono-(N-formylmethyl)-amidediethylacetal by FAB-MS. The second HPLC fraction was identified byFAB-MS, through strong signals at m/z 624, 646 and 662 (protonatedmolecular ion, sodium-cationized ion and potassium-cationized ion,respectively), as the dimer resulting from the acylation of twomolecules of aminoacetaldehyde by the bisanhydride(DTPA-bis-(n-formylmethyl)-amide-diethylacetal).

(b) Preparation ofdes-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine-methylester/DTPA-mono-(N-formylmethyl)-amideConjugate

The acetal protecting group DTPA-mono-(N-formylmethyl)-amidediethylacetal was removed quantitatively with 1 M HCl. 0.63 mg ofdeprotected material was dissolved in 250 μl of a buffer made byadjusting the pH of 1% (v/v) aqueous acetic acid to pH 3.5 with 1 MNaOH. 0.2 mg ofdes-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine-methylester seeExample 4(a)) was dissolved in 9 μl of theDTPA-mono-(N-formylmethyl)-amide solution and 10 μl of aqueous sodiumcyanoborohydride (10 mM) were added. After 90 min. at room temperature,the product, which precipitated, was isolated by acidification (torender it soluble) followed by HPLC on the C-18 cartridge. While theproduct eluted too close to the position of the starting insulinderivative for any useful separation to be attempted, electrophoresis oncellulose at pH 8 showed that a coupling yield of about 40% had beenachieved.

Example 11 Preparation of a Dimer of Insulin Usingdes-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine-methylester andm-benzene-dialdehyde

To 100 μl of 1 M propionic acid containing 2 mgdes-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine-methylester and 26.8μg of m-benzene-dialdehyde were added 100 μl of 1 M propionic acidcontaining 62.8 μg sodium cyanoborohydride. After 5 min. at roomtemperature, the product was isolated by HPLC on a C-18 cartridge. Gelfiltration on SEPHADEX G-50 showed that the starting insulin derivativehad been transformed, in a yield of about 70%, to a dimeric compound(based on elution from SEPHADEX G-50) which eluted from the C-18cartridge (analytical run, linear gradient of 25-40%, v/v, acetonitrilein 0.1% trifluoroacetic acid at 1% per min.) appearing three minutesafter the starting material. Under similar reaction conditions,unmodified porcine insulin produces less than 1% dimeric material. Thestructure of the dimeric product is:

Example 12 Preparation of ades-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine/des-Ala^(B30)-insulin-B29-m-formylanilideConjugate

10.2 mg of des-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine-methylester(prepared as described in Example 4(a)) was saponified in 5 ml ofaqueous 1% (w/v) ammonium bicarbonate solution brought to pH 9.5 withNaOH. After incubation at 37° C. for 24 h, the saponified product wasisolated on a Sep-pak according to the manufacturer's instructions,eluting with 50% (v/v) aqueous acetonitrile which was 0.05% intrifluoroacetic acid. Saponification of the methylester was confirmed byelectrophoresis on cellulose-acetate at pH 8. The product was recoveredby lyophilization after removal of the acetonitrile on the rotaryevaporator at room temperature. To one volume of a solution ofdes-Ala^(B30)-insulin-B29-p-amino-L-phenylalanine (50 mg/ml in 1 Mpropionic acid) was added one volume of a solution ofdes-Ala^(B30)-insulin-B29-m-formylanilide (prepared according to Example1(a) with purification by HPLC; also 50 mg/ml in 1 M propionic acid) andhalf a volume of a solution of sodium cyanoborohydride (3.14 mg/ml) in 1M propionic acid). After 4 min., it was shown by HPLC (C-18 cartridge)that about 70% of the starting protein has been transformed to a morehydrophobic material eluting two minutes later than the starting amineand having the properties of a dimer of presumed structure:

Example 13 (a) Preparation of N-hydroxysuccinimide Ester of4-methoxy-3-nitrobenzoic Acid

1.97 g (10 mmoles) of 4-methoxy-3-nitrobenzoic acid was dissolved in 50ml of acetonitrile. 1.15 g (10 mmoles) N-hydroxysuccinimide was addedwith agitation. Finally 2.063 g (10 mmoles) ofN,N′-dicyclohexylcarbodiimide were added and the mixture agitated. After2 hours at approx. 20° C. a precipitate of dicyclohexyl-urea had formed.The solution was filtered through a Gooch 3G funnel. The formation ofester was determined by t.l.c. of the filtrate (CHCl₃/MeOH=9:1, v/v).Observation under U.V. revealed the ester as a dark spot of R_(f)approx. 0.9. After evaporation of the acetonitrile a whitish-yellowpowder remained in the flask. The powder was redissolved in 50 ml of hotisopropanol. Approximately 2 spatula-tips activated charcoal were addedand the solution was boiled in a boiling water-bath for about 5 minutes.The boiling solution was quickly poured through a sintered glass filter(G4, previously heated by a passage of hot isopropanol) into a hotflask. The solution was allowed to cool down overnight. White,needle-like crystals were formed (m.p. approx. 145-152° C.) having theexpected t.l.c. properties.

(b) Insulin Activation with N-hydroxysuccinimide Ester of4-methoxy-3-nitrobenzoic Acid

61 mg (10 μmole) of insulin (porcine) were dissolved in 2.1 ml of DMSO.To this were added 900 μl of a solution of N-ethyl-morpholine-carbonate,pH 8.3 (this solution was prepared freshly from 10 ml ofN-ethyl-morpholine and powdered dry ice with stirring until pH of thesolution, as verified on an electrode, was 8.3).

A solution of 3.6 mg of the N-hydroxysuccinimide ester of4-methoxy-3-nitrobenzoic acid was made in 100 μl of DMSO. This was addedto the above insulin solution with agitation. The reaction mixture wasallowed to stand at ambient temperature for 20 minutes. The ratio ofN-hydroxysuccinimide ester to insulin was 1:1 on a molar basis. Thereaction was stopped by acidification, i.e. addition of 60 μl HCl, 37%(pH of reaction solution approx. 2.5 on paper). The acidified reactionsolution was dialysed against NH₄HCO₃ (1%), then lyophilized.

The nitro-benzoyl-insulin derivative was purified by ion-exchange on a11×2.5 cm DEAE-A 25 column (flow rate approx. 1 ml/min, equilibrated in0.1 M tris-HCl/7 M urea, pH 8.4, elution with a 0 to 0.15 M NaClgradient). The peak fractions were pooled, dialysed first against waterthen against NH₄HCO₃ (1%, w/v) and then lyophilized. Each pool wasanalysed by HPLC (same conditions as before) to confirm the purity ofthe monosubstituted nitrobenzoyl-insulin.

(c) Reduction of Monosubstituted Nitrobenzoyl-insulin to MonosubstitutedAminobenzoyl-insulin

5 mg of purified monosubstituted-nitrobenzoyl-insulin was dissolved in 1ml tris-HCl (50 mM) pH 8.3 buffer. 60 μl of a sodium dithionite solution(50 mM in H₂O) was added to it (3.6 excess dithionite over insulin).After agitation on a Vortex mixer the solution was allowed to stand atambient temperature (approx. 20° C.) for 3 minutes.

The reaction was stopped by diluting the solution to 4 ml with 50 mMtris-HCl pH 8.3 buffer. The reaction solution was desalted by passagethrough a SEP-PAK cartridge (WATERS). The sample was adsorbed onto thecartridge with a solution of 10% CH₃CN/0.1% TFA and eluted off with asolution of 40% CH₃CN/0.1% TFA. The excess acetonitrile was dried offunder a stream of compressed air, then the solution was lyophilized.

The resultant aminobenzoyl-insulin powder was purified by preparativeHPLC (RP-18 column; buffer: 0.3 M (NH₄)₂SO₄, pH 2.7; load at 24% CH₃CN,linear gradient up to 35% CH₃CN over 45 minutes). The peak fractionswere pooled separately, the excess acetonitrile was evaporated under astream of compressed air and the remaining solutions were thenlyophilized.

The peaks that emerged at or near the expected position foraminobenzoyl-substituted insulin were tested as follows. Thefreeze-dried pool was dissolved to 7.9 mg/ml in 10 nM HCl and trialSchiff-base couplings to benzaldehyde were carried out in the usual way(10 mM, aqueous, solubilised with a little solid urea). The pools whichwere judged by HPLC to have coupled most effectively with thebenzaldehyde solution were selected for subsequent tests. Two peaks,presumably isomers, were seen to couple very well with benzaldehyde.

(d) Preparation ofAminobenzoyl-insulin/des-Ala^(B30)-insulin-B29-m-formylanilide Conjugate

Method:

The purified insulin-aryl-NH₂ (aminobenzoyl-insulin) pool was coupled toinsulin-aryl-CHO (des-Ala^(B30)-insulin-B29-m-formylanilide, prepared asdescribed in Example 1(a)) using the following method:

  Insulin-aryl-NH₂ (6.8 mg/ml)   5 μl (34 μg or 5.7 nmoles) +Insulin-aryl-CHO (7.8 mg/ml) 4.3 μl (34 μg or 5.7 nmoles) + NaBH₃ CN (3mM)   2 μl (6 nmoles) + Acetic acid 4.17% adjusted to 1.2 μl pH 3.5 withstrong NaOH

After vortexing and leaving at ambient temperature for 30 minutes, thesolution was analysed by HPLC as before and by a 15-25% SDS gradient gel(at pH 8.3). Both HPLC and the SDS gel showed formation of coupledmaterial. On the gel a band of MW approx, 11,900 was observed confirmingthe presence of a dimer of insulin. cl Example 14

(a) Preparation of Fab-p-aminophenylalanine Amide with CarboxypeptidaseY

A solution of an Fab antibody fragment was prepared by papain digestionof an antibody, gel-filtration chromatography, and concentration bymembrane filtration using standard methods. The solution was 4.3 mg/mlin protein, and the solvent was Dulbecco phosphate-buffered saline. To70 μl of this solution was added 7 mg of solid p-aminophenylalanineamide, freeze dried from a solution that had been adjusted to pH 8.5with 0.01M NaOH or 0.01M HCl as necessary. Once thep-amino-phenylalanine amide was in solution, 7 μl of carboxypeptidase YCarlsberg (1 mg solid corresponding to 0.106 mg protein, per 50microliters) was added. After two and a half hours at room temperature,the enzyme was inhibited with PMSF (7 microliters of a 10 mg/ml solutionin acetonitrile) and left at 0° C. for 10 minutes. The digest was thendiluted with 500 microliters of Dulbecco phosphate-buffered saline andsubjected to gel filtration (SEPHADEX G50; column 60 cm×0.9 cmdiameter). The protein peak was collected and concentrated to 1.1 mg/ml(monitored by O.D.₂₈₀; O.D. 1.3=1 mg/ml).

(b) Preparation of Fab-p-aminophenylalanine Amide with Papain

To one volume of a solution of an Fab fragment (the same solutionconditions as that used in (a) above) were added two volumes of asolution of 1M p-aminophenylalanine amide (pH adjusted to pH 6.7 withacetic acid). Three volumes of butane-1,4-diol were added, followed by0.06 volumes of cysteine (1M). A 50:50 mixture of papain suspension andbutane-1,4-diol was prepared (final papain content 18 mg/ml) and 0.28volumes of the mixture were added at once to the Fab solution. Theapparent pH was checked with pH paper: if the apparent value was lowerthan pH 6.2 it was raised to 6.2-6.7 with 0.01M NaOH (with careful,rapid mixing). After 18 hours at room temperature the papain wasinactivated by addition of the appropriate volume of iodoacetic acid,0.5M, brought to pH 7.0 with 1% NaHCO₃ solution. After incubation for 10minutes at room temperature the protein conjugate was isolated onSEPHADEX G50 as described above.

(c) Preparation of Fab-p-aminophenylalanine Amide with Trypsin

To one volume of a solution of an Fab fragment (the same solutionconditions as in (a) were added two volumes of the solution of 1Mp-aminophenylalanine amide pH 6.7 used in (b). Butane-1,4-diol (3.2volumes) was then added, followed by 0.2 volumes of bovine trypsin (10mg/ml in HCl 10⁻²M). After 18 hours at room temperature the pH wasbrought to 3.5 with acetic acid, and the modified Fab isolated by gelfiltration as above.

(d) Preparation of N^(ε)-dansyl-N^(α)-(m-formylbenzoyl)-lysine

4—Carbobenzaldehyde (Fluka) was stirred with dry methanol at roomtemperature for 24 hours. T.l.c. on silica with chloroform/methanol(9:1, v/v) showed essentially quantitative conversion to the acetal, asjudged by inspection after spraying with a saturated solution of2,4-dinitrophenylhydrazine in 2M HCl (Rf of product approx. 0.3, Rf ofstarting aldehyde approx. 0.15). The product was recovered by rotaryevaporation without heating and traces of water were removed from thewhite cake by the addition of 50 ml dichloromethane followed by rotaryevaporation once again. To the cake were added, with mixing, 2.3 g ofN-hydroxysuccinimide in 120 ml. ethyl acetate, followed by 4.12 gdicyclohexylcarbodiimide in 20 ml ethylacetate. The quantitativetransfer of the carbodiimide from the weighing tube to the reactionvessel was ensured by a wash with 10 ml ethylacetate. A heavyprecipitate formed after about 1 minute. After one hour at roomtemperature, the precipitate was removed by filtration. The precipitatewas washed with 20 ml ethylacetate and the combined filtrates wererotary evaporated to an oil, which spontaneously crystallised.Recrystallisation from 120 ml propan-2-ol gave 2.75 g of a whitecrystalline product. Mass spectrometry (fast-atom bombardment) showed astrong signal at m/z 262, interpreted as M+H⁺-MeOH. The compound gives asingle spot on t.l.c. (the same chromatographic system and spray asabove) with an Rf of about 0.6.

N^(ε)-dansyl-L-lysine (Sigma, 9.5 mg) was dissolved in 100 μldimethylformamide under gentle warming. N-ethylmorpholine (3 μl) wasadded, so that the apparent pH, as judged by spotting onto moist pHpaper, was between 8 and 8.5. This solution was mixed with a solution of7.3 mg of the hydroxysuccinimido ester of 4-carboxybenzaldehydedimethylacetal in 50 μl dimethylformamide. The apparent pH was checkedin the same way as before, and adjusted with N-ethylmorpholine (1 μl ata time) if necessary. After 4 hours and 30 minutes (the apparent pHhaving been checked from time to time and adjusted if necessary), asimilar solution of 7.3 mg of the active ester was added and thereaction allowed to continue for a further 4 hours. The reaction, asjudged by t.l.c., was then essentially complete. The t.l.c. system wasthat described in Example 1(c) and the Rf values were approx. 0.3 and0.6 for dansyl-lysine and the product, respectively. The mixture wasthen diluted to 1 ml with acetic acid (1%, v/v), centrifuged, and thesupernatant subjected to HPLC (system of Example 4(c)). The majorfluorescent peak eluting on the gradient (around 40% acetonitrile) waspooled, and the acetonitrile was driven off in a current of air. Theproduct was characterized by measurement of its electrophoretic mobilityat pH 6.5 (predicted and observed, 0.5 [mAsp=1.0]). To this stocksolution (600 μl) were added 6 μl M HCl, and the solution was dilutedten-fold with 10⁻³M HCl. This brought the concentration to 0.7 mM, asjudged by direct comparison of the u.v. absorption spectrum with that ofa solution of dansyl-lysine of known concentration. It was assumed thatthe optical density of the dansyl chromophore would be approximately thesame in the two substances.

It was expected that the acetal protection would be removed during HPLC(pH of the system approx. 2). This hypothesis was confirmed by the factthat the product underwent rapid coupling to aromatic amines via theSchiff-base/cyanohydride reaction without the need for any prior acidtreatment.

(e) Coupling between Fab-p-aminophenylalanine Amide andN^(ε)-dansyl-N^(α)-(m-formylbenzoyl)-lysine

The Fab derivative obtained in acccordance with procedure (a) (1.1mg/ml) was buffered at either pH 3.5 (acetate buffer made by adjusting12% (v/v) acetic acid to pH 3.5 with concentrated NaOH and then dilutingto a final equivalent of 10% acetic acid) or pH 2 (1M propionic acidbrought down to pH 2 by 1M HCl). In either case 2 volumes of buffer wereused for every five volumes of Fab solution. To this solution was added1 volume of a solution of the lysine derivative obtained in accordancewith procedure (d) (approx. 0.7 mM in 10⁻³M HCl) and 1 volume of sodiumcyanoborohydride (3 mM). In controls, the cyanoborohydride was replacedby water. Samples were withdrawn from time to time and the extent of thecoupling reaction was judged by cellulose-acetate electrophoresis. Saltand any uncoupled aldehyde were largely eliminated before running byacetone precipitation of the protein, which was taken up forelectrophoresis in the electrophoresis buffer (2% formic acid/8% aceticacid/8M urea). The coupling appeared to reach its maximum between 20 and80 minutes. The fluorescent protein conjugate was isolated byprecipitation (cold acetone) and washing. Controls showed nofluorescence.

Example 15 Preparation of m-aminobenzoyl-polyglutamic Acid Substitutedon the Side Chains with Ferrioxamine B

Polyglutamic acid (Sigma Inc., glutamic acid polymerised by peptide-bondformation through its alpha amino and carboxyl groups, average number ofresidues per chain approx. 50) was suspended in dimethylsulphoxide (100mg in 1 ml DMSO). The apparent pH, as judged externally with damp pHpaper, was brought to 8 by cautious addition of N-methyl-morpholine.Hydroxysuccinimido ester of m-aminobenzoic acid (Example 1) was thenadded (75 mg). The apparent pH was readjusted to 8 withN-methyl-morpholine. The polyglutamic-acid suspension gradually clearedover the course of 18 hours at 20° C. Paper electrophoresis showed thatthe amino group had essentially fully reacted after 24 hours. Water (5ml) was then added and the solution allowed to stand for 1 h at 20° C.,in order to hydrolyse any remaining active ester (the pH remained above7 during the period). A precipitate which formed as soon as the waterwas added was removed at the end of the 1 hour's period bycentrifugation. The supernatant was adjusted to 5 with acetic acid (10%,v/v, previously adjusted to pH 3). The solution was stored and worked upas required in lots equivalent to 5-10 mg of polyglutamic acid. Thework-up consisted of adsorption on a Sep-Pak C₁₈ cartridge (type 51910)that had been equilibrated with HCl, 10⁻⁴M. The wash was with 2×10 ml ofthe same HCl solution, and desorption with 2 ml 10⁻⁴M HCl/acetonitrile(6:4, v/v). The acetonitrile was then removed in a current of air, andthe turbid suspension of the polyglutamic acid derivative was driedunder reduced pressure. This material was coupled to ferrioxamine B asfollows.

To 1 volume of a solution of the polyglutamic acid derivative (20 mg/mlin dimethylformamide) were added 1.8 volumes of a solution of1,1-carbonyl-diimidazole (80 mg/ml DMF). After 30 minutes at 20° C.solid ferrioxamine B was added (1 mg/14 μl of reaction mixture). Thegreat majority of the ferrioxamine dissolved at once. After 30 minutesthe reaction mixture was diluted with 5 volumes of acetic acid (0.1%,aqueous) and applied to SEPHADEX G50. The polymer peak was concentratedon SEP PAK as described above, except that the equilibration solutionwas 0.1% aqueous CF₃COOH, the wash was 0.1% aqueousCF₃COOH/acetonitrile, 19:1 (v/v) and the desorption took place in 0.1%aqueous CF₃COOH/acetonitrile, 1:4 (v/v). After removing the acetonitrilefrom the desorbed fraction in a current of air, the aqueous solution wasapplied for HPLC (C₁₈ reversed-phase) in 0.1% CF₃COOH, with a gradientof 0-100% acetonitrile in 30 minutes. The wanted product eluted as arelatively broad peak at around 50% acetonitrile. The peak fractionswere dried in the usual way. The BOC protection was removed by anhydrousCF₃COOH (50 μl per mg of product for 30 minutes at 20° C.). Spectroscopyand amino-acid analysis showed an incorporation of between 0.71 and 0.76residues of ferrioxamine B per residue of glutamic acid.

Example 16 (a) Preparation of the Chelon H₂N—O—CH₂—CO-ferrioxamine B

1.093 g O-carboxymethyl-hydroxylamine-hemihydrochloride (Fluka) wasN-protected by introduction of the tert-butyloxycarbonyl group understandard conditions (water/methanol) using 4.365 g Boc₂O (Fluka). The pHwas maintained at 9 with NaOH. After 16 hours at 22° C., the solutionwas evaporated and the solid residue taken up in 10 ml of water. Aftercooling to 0° C., the solution was acidified carefully to pH 3 and theprecipitate which formed was collected by centrifugation. Yield afterdrying under high vacuum: 1 g. The product was identified asBoc-NH—O—CH₂—COOH by negative ion FAB/MS (intense M−H at m/z 190).

191 mg of Boc-NH—O—CH₂—COOH were dissolved in 25 ml DMSO and 115 mg ofN-hydroxysuccinimide were added. To the resulting solution were added210 mg of dicyclohexylcarbodiimide dissolved in 5 ml DMSO and thereaction mixture was left at room temperature overnight. A solution offerrioxamine B in DMSO (6.6 ml, 15 mg/ml) was added and sufficientN-methyl-morpholine to bring the pH (externally measured with moistMerck pH strip having chemically bound dyes to permit rinsing with waterto remove the colour due to ferrioxamine) to 8. After 4 hours at roomtemperature the reaction mixture was diluted to about 150 ml with waterand the N,N′-dicyclohexylurea precipitate removed by centrifugation. Thesolution was acidified to pH 3 with acetic acid and the product wasrecovered, in portions, on a C18 Sep-Pak column as follows. After aninitial methanol wash, the Sep-Pak column was equilibrated with 0.1%acetic acid, a portion of the sample applied, and the column washed with0.1% acetic acid to remove DMSO. Washing with 0.1% aceticacid/acetonitrile (9:1, v/v) removed traces of unreacted ferrioxamine B,and the wanted product was eluted with 0.1% acetic acid/acetonitrile(7:3, v/v). After pooling of the product portions, solvent was removedby rotary evaporation and the Boc group removed by dissolving theproduct in 1 ml trifluoroacetic acid and incubating at 22° C. for 45minutes. The acid was removed by rotary evaporation, whereupon theresidue was taken up in water and purified in a Sep-Pak column asdescribed above, except that this time, since the hydrophobic Boc grouphad been removed, the colour was eluted with 0.1% aceticacid/acetonitrile (9:1, v/v). Acetonitrile was removed by rotaryevaporation and the aqueous solution was then freeze-dried: yield about35 mg. The product ran as an orange-brown, ninhydrin-negative, singlespot on silica t.l.c. in butanol/acetic acid/water/acetone (7:2:4:7,v/v) with an Rf of about 0.35 (Rf ferrioxamine about 0.1), it ran as asingle spot a little slower than ferrioxamine on paper electrophoresisat pH 1.9, and was identified as NH₂—O—CH₂—CO-ferrioxamine B by positiveion FAB/MS (intense M+H at m/z 687).

(b) Coupling of the Chelon H₂N—O—CH₂—CO-ferrioxamine B to Aldehydes andKetones

In order to show that the new chelon, NH₂—O—CH₂—CO-ferrioxamine B,reacts under mild conditions with aldehydes and ketones, the chelon(about 5 mM) was incubated respectively with 4-carboxybenzaldehyde(about 1.3 mM), with o-aminobenzaldehyde (about 3 mM), with3-aminoacetophenone (about 3 mM), with N-acetyl-3-aminoacetophenone(about 3 mM), with 4-aminoacetophenone (about 3 mM), withN-acetyl-4-aminoacetophenone (about 3 mM), with heptanaldehyde (about 3mM), with nonan-5-one (about 3 mM), and with pyruvate (about 3 mM), inacetate buffer at pH 3, 4, 5, and in pyridine acetate buffer at pH 6.5,at 22° C. Control incubations were performed with ferrioxamine B undersimilar conditions. With the hydroxylamino chelon, rapid reaction ensuedwith all reagents at pH 3 and 4, to give a coloured product morehydrophobic than the chelon in the t.l.c. system described above, and inthe expected ratio in view of the excess of chelon. The spots did nottrail, and no further reaction occured over at least 24 hours. The morereactive reagents (pyruvate and aldehydes except o-aminobenzaldehyde)reacted within 5 hours up to pH 6.5. Under similar conditions,unsubstituted ferrioxamine, which has an aliphatic amino group, showedno sign of reaction with any of the reagents: any products which mayhave been formed by temporary association of carbonyl compound withferrioxamine must have been unstable to the conditions of analysis(t.l.c. in butanol/acetic acid/water/acetone). The oximes obtained bythe coupling are stable compounds.

(c) Coupling of desAla^(B30)-insulin-B29-m-formanilide toH₂N—O—CH₂—CO-ferrioxamine B

1 mg of desAla^(B30)-insulin-B29-m-formanilide (obtained according toExample 1a) was dissolved in 100 μl of a solution ofNH₂—O—CH₂—CO-ferrioxamine B (10 mM in 0.1% acetic acid) and incubated at22° C. for 2 hours. This led to expected formation of the O-alkyloximeof the protein derivative, which was detected by t.l.c. on silica sheetsusing butanol/acetic acid/water/acetone (7:2:4:7, v/v), which showed thepresence of a coloured spot of almost zero mobility, staining positivewith cadmium-ninhydrin. This material was shown to be the expectedconjugate, desAla^(B30)-insulinyl-3-aminobenzaldehyde-O-alkyloxime, byreversed phase HPLC (using 0.1% TFA and acetonitrile, a Macherey-Nagel 5μm C8 300 A 4 mm×25 cm column at a flow rate of 1 ml/min; the proteinderivative, now coloured, elutes close to the position ofdesAla^(B30)-insulinyl-3-aminobenzaldehyde, but well separated fromstarting material, on a gradient of 1% acetonitrile per minute) and byelectrophoresis on cellulose acetate at pH 8.0 (the coloured proteinderivative ran in a position characteristic of insulin derivativeshaving lost one negative charge, and stained red with Ponceau S).

Example 17

3-Aminopropane-1,2-diol (0.5M) was coupled to desAla^(B30)-insulin (4mM) in 90% butane-1,4-diol at pH 6.5 (uncorrected glass electrode, pHadjusted with acetic acid) within 2 hours at 22° C. using TPCK-treatedbovine trypsin (enzyme/substrate ratio 1:10, w/w) as catalyst. Thereaction was very clean and the coupling yield as judged byelectrophoresis on cellulose acetate was about 70%. A procedure using1,3-diaminopropane-2-ol (0.5M) in place of 3-aminopropane-1,2-diol gavea similar result.

The desAla^(B30)-insulin-B29-derivatives are susceptible to periodateoxidation under mild conditions yielding a deriva- tive with a carboxyterminal aldehyde group.

Example 18 (a) Preparation of desAla^(B30)-insulin-B29-m-acetanilide

10 mM desAla^(B30)-insulin in 90% butane-1,4-diol was coupled to3-aminoacetophenone (0.5M) at a pH (uncorrected glass electrode) of 5.5(pH adjusted with acetic acid) for a time of 5 hours at 22° C. usingTPCK-treated bovine trypsin (enzyme/substrate ratio 1:10, w/w) ascatalyst. The reaction was very clean as judged by electrophoresis oncellulose acetate and the coupling yield was about 70%. The reaction wasquenched with acetic acid and the mixture applied to a column ofSephadex G50 fine (2.6 cm×90 cm), equilibrated and eluted with 1% aceticacid. This step removed trypsin and small molecules, including excessreagent. The insulin peak was lyophilised (yield 8.8 mg) and shown to bea mixture of coupled and uncoupled product (coupled product about 70%)by electrophoresis on cellulose acetate at pH 8.0. Coupled product wasseparated from uncoupled product by reversed phase HPLC (using 0.1% TFAand acetonitrile, a Macherey-Nagel 5 μm C8 300 A 4 mm×25 cm column at aflow rate of 1 ml/min; the coupled product elutes later, well separatedfrom desAla-insulin on a gradient of 1% acetonitrile/min.

(b) Coupling of desAla^(B30)-insulin-B29-m-acetanilide toH₂N—O—CH₂—CO-ferrioxamine B

A portion of the product obtained (0.8 mg) was incubated at roomtemperature in solution in 100 μl 0.1% acetic acid with 10 mM ofNH₂—O—CH₂—CO-ferrioxamine B. After a few hours, t.l.c. (on silica sheetsusing butanol/acetic acid/water/acetone, 7:2:4:7, v/v) showed presenceof a coloured spot of almost zero mobility, staining positive withcadmium-ninhydrin. This material was shown to be the expected conjugate,desAla^(B30)-insulin-B29-m-acetyl-anilide/H₂N—O—CH₂—CO-ferrioxamineB-oxime by reversed phase HPLC on the same Macherey-Nagel column (thecoupled protein derivative, now coloured, elutes close to the positionof desAla^(B30)-insulin-B29-m-acetanilide) and by electrophoresis oncellulose acetate at pH 8.0 (the coloured protein derivative runs in aposition characteristic of insulin derivatives having lost one negativecharge, and stains red with Ponceau S).

Example 19 (a) Preparation of Porcine [glyoxyloyl^(A1)]insulin and[glyoxyloyl^(A1), benzyloxalyl^(B1)]insulin

12 mg of porcine insulin were dissolved in 12 ml of a solutioncontaining 2M pyridine, 0.8M acetic acid, 10 mM sodium glyoxylate and 2mM CuSO₄ (pH 5.5). The reaction was essentially complete after 20minutes but was allowed to continue for 3 hours. Samples takensubsequent to 20 minutes showed on reversed phase HPLC (same system asin Example 18a) little starting insulin, some material eluting at theposition of a mono-transaminated species ([glyoxyloyl^(A1)]insulin), andthe great majority at the position of a di-transaminated species([glyoxyloyl^(A1), benzyloxalyl^(B1)]insulin). The main reaction mixturewas diluted to 50 ml with water and passed through a Waters Sep-Pakcolumn, which was then washed with 20 ml of 0.1% aqueous trifluoraceticacid/acetonitrile (9:1, v/v). The modified insulin was then eluted with2 ml of 0.1% trifluoracetic acid (aqueous)/acetonitrile (2:3, v/v). Theacetonitrile was removed in a current of air and the resulting solutionwas lyophylized.

(b) Coupling of Porcine [qlyoxyloyl^(A1)]insulin and [glyoxyloyl^(A1),benzyloxalyl^(B1)]insulin to H₂N—O—CH₂—CO-ferrioxamine B

The material obtained according to (a) was coupled toH₂N—O—CH₂—CO-ferrioxamine B by the identical method described in Example18b. HPLC permitted the isolation of two coloured protein derivatives,one eluting near the position of the mono-transaminated insulin, and theother (the majority) eluting near the position of the di-transaminatedderivative.

Example 20

Ribonuclease S-protein was isolated from ribonuclease S (Sigma ChemicalCo.) by reversed phase HPLC on the Beckman machine using the previouslymentioned (Example 18a) Macherey-Nagel column and a gradient ofacetonitrile, in 0.1% trifluoroacetic acid: the S-protein elutes laterand well-separated from the S-peptide, and the elution positions of bothS-peptide and S-protein were verified by running authentic standards(obtained from Sigma). The purified S-protein obtained from 5 mg ofribonuclease S, dissolved in 1.2 mM HCl, was buffered at pH 7 withimidazole and the sample divided into two 5 ml aliquots. One portion wasoxidised with 5 μl of a solution of periodic acid (19.2 mg/ml in water)at 22° C. for 6 minutes. After this time, the reaction was quenched with1 ml ethane-1,2-diol. The reaction mixture was applied to a SEP PAK C18cartridge equilibrated with 1.2 mM HCl. After washing with 1.2 mM HCl,the protein fraction was eluted with 3 ml 1.2 mM HCl/acetonitrile (4:6,v/v). To half of the eluate was added 1.5 ml NH₂—O—CH₂—CO-ferrioxamine B(10 mM in 50 mM sodium acetate, pH 5; after mixing 5.1). The sample wasincubated overnight at 22° C. Upon preparative HPLC, a coloured,protein-containing material was eluted from the Macherey Nagel column(under conditions similar to those used to isolate the S-protein) closeto the position of S-protein: the material still contained someuncoupled protein. The presence of the ferrioxamine chromophore in thematerial isolated by HPLC was confirmed by spectrophotometry on a VarianCary instrument.

What we claim is:
 1. A composition containing conjugated derivatizedprotein or polypeptide molecules, or salts thereof, wherein saidconjugated derivatized protein or polypeptide molecules of saidcomposition consist essentially of a compound selected from the formula:A—X—Z—X′—B wherein: A is a protein or polypeptide, and wherein A has acarboxy and amino terminus and is conjugated to X—Z—X′—B only at eithersaid carboxy terminus of A or at said amino terminus of A; B is apolymeric compound; X and X′ independently from each other are bivalentorganic radicals or independently from each other are present or absent;Z is a bivalent radical selected from the group consisting of: —C(R)═N—,—N═C(R)—, —CH(R)—NH—, —NH—CH(R)—, —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)—, —NH—CH(R)—Y—CH(R)—NH—, —C(R)═N—O—, —O—N═C(R)—,—CR(R)—NH—O—, —O—NH—CH(R)—, —C(R)═N—O—Y—O—N═C(R)—,—O—N═C(R)—Y—C(R)═N—O—, —CH(R)—NH—O—Y—O—NH—CH(R)— and—O—NH—CH(R)—Y—CH(R)—NH—O—; where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group and Y is abivalent organic group; wherein said conjugated derivatized protein orpolypeptide molecules of said composition are produced via formation ofa Schiff base, hydrazone, oxime or azomethine compound that results inthe presence of at least one radical having the formula —C(R)═N—, or—N═C(R)—, or, when reduced, —CH(R)—NH— or —NH—CH(R)—.
 2. The compositionof claim 1, wherein said polymeric compound B is a protein orpolypeptide that is the same or different from said protein orpolypeptide A, or is a reporter group or cytotoxic agent; and wherein Zis a bivalent radical selected from the group consisting of: —C(R)═N—,—N═C(R)—, —CH(R)—NH—, —NH—CH(R)—, —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)— and —NH—CH(R)—Y—CH(R)—NH—.
 3. The composition ofclaim 1, wherein said polymeric compound B is a protein or polypeptidethat may be the same or different from said protein or polypeptide A, oris a reporter group or cytotoxic agent; and wherein Z is a bivalentradical selected from the group consisting of: —C(R)═N—O—, —O—N═C(R)—,—CH(R)—NH—O—, —O—NH—CH(R)—, —C(R)═N—O—Y—O—N═C(R)—,—O—N═C(R)—Y—C(R)═N—O—, —CH(R)—NH—O—Y—O—NH—CH(R)— and—O—NH—CH(R)—Y—CH(R)—NH—O—.
 4. The composition of claim 2, wherein R ishydrogen.
 5. The composition of claim 3, wherein R is hydrogen.
 6. Thecomposition of any of claim 2, 3, 4 or 5, wherein A is connected to—X—Z—X′—B, at the carboxy terminus of A.
 7. The composition of any ofclaim 2, 3, 4 or 5, wherein A is connected to —X—Z—X′—B, at the aminoterminus of A.
 8. The composition of claim 6, wherein R is hydrogen. 9.The composition of claim 7, wherein R is hydrogen.
 10. The compositionof any of claim 2 or 4, wherein Z is —C(R)═N—, or —N═C(R)—.
 11. Thecomposition of any of claim 3 or 5, wherein Z is —C(R)═N—O— or—O—N═C(R)—.
 12. The composition of claim 2, wherein Z is —C(R)═N—, or—N═C(R)—.
 13. The composition of claim 12, wherein A is connected to—X—Z—X′—B, at the carboxy terminus of A.
 14. The composition of claim12, wherein A is connected to —X—Z—X′—B, at the amino terminus of A. 15.The composition of claim 13 or 14, wherein R is hydrogen.
 16. Thecomposition of claim 2, wherein Z is —C(R)═N—O—, or —O—N═C(R)—.
 17. Thecomposition of claim 16, wherein A is connected to —X—Z—X′—B, at thecarboxy terminus of A.
 18. The composition of claim 16, wherein A isconnected to —X—Z—X′—B, at the amino terminus of A.
 19. The compositionof claim 17 or 18, wherein R is hydrogen.
 20. The composition of claim2, wherein Z is —CH₂—NH—, or —NH—CH₂—.
 21. The composition of claim 20,wherein A is connected to —X—Z—X′—B, at the carboxy terminus of A. 22.The composition of claim 20, wherein A is connected to —X—Z—X′—B, at theamino terminus of A.
 23. The composition of claim 21 or 22, wherein R ishydrogen.
 24. The composition of claim 3, wherein Z is —C(R)═N—O—, or—O—N═C(R)—.
 25. The composition of claim 24, wherein A is connected to—X—Z—X′—B, at the carboxy terminus of A.
 26. The composition of claim24, wherein A is connected to —X—Z—X′—B, at the amino terminus of A. 27.The composition of claim 25 or 26, wherein R is hydrogen.
 28. Thecomposition of claim 2, wherein Z is —CH═N—Y—N═CH—, —N═CH—Y—CH═N—,—CH2—N—Y—N—CH2—, or —NH—CH2—Y—CH2—NH—.
 29. The composition of claim 28,wherein A is connected to —X—Z—X′—B, at the carboxy terminus of A. 30.The composition of claim 28, wherein A is connected to —X—Z—X′—B, at theamino terminus of A.
 31. The composition of claim 29 or 30, wherein R ishydrogen.
 32. The composition of claim 1, wherein Z is selected from thegroup consisting of: —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)—, and —NH—CH(R)—Y—CH(R)—NH—; and at least one of X,X′ or Y comprises an aromatic group directly adjacent to an N atom of Z.33. The composition of claim 32, wherein Y is a phenylene radical. 34.The composition of claim 32, wherein X is a phenylene radical.
 35. Thecomposition of claim 32, wherein X′ is a phenylene radical.
 36. Thecomposition of claim 1, wherein said protein or polypeptide A isconjugated to X—Z—X′—B only at said carboxy terminus of A, wherein saidderivatized protein or polypeptide is prepared using reverse proteolysisto attach an aldehyde, aldehyde precursor, or hydrazide to said carboxyterminus followed by said formation of a Schiff base, hydrazone, oximeor azomethine compound.
 37. The composition of claim 1, wherein saidprotein or polypeptide A is conjugated to X—Z—X′—B only at said carboxyterminus of A, wherein X comprises a reactive amino group and whereinsaid derivatized protein or polypeptide or salt thereof is preparedthrough a reaction in which said reactive amino group is directlyattached to an aromatic group, to the oxygen of an O-alkylatedhydroxylamine, or to the nitrogen of the —NH—CO— moiety of a hydrazide.38. The composition of claim 1, wherein said protein or polypeptide orsalt is conjugated exclusively at its amino terminus with the same or adifferent protein or polypeptide, wherein R is hydrogen.
 39. Thecomposition of claim 1, wherein said protein or polypeptide A is animmunoglobulin or a fragment thereof that functions as animmunoglobulin.
 40. The composition of claim 39, wherein saidimmunoglobulin is an IgG molecule or a fragment thereof that functionsas an immunoglobulin.
 41. The composition of claim 39, wherein saidprotein or polypeptide is a Fab or F(ab′)₂ fragment of animmunoglobulin.
 42. A composition containing conjugated derivativeprotein or polypeptide molecules, or salts thereof, wherein saidconjugated derivatized protein or polypeptide molecules of saidcomposition consist essentially of a compound selected from the formula:A—X—Z—X′—B wherein: A is a protein or polypeptide, and wherein A has acarboxy and amino terminus and is conjugated to X—Z—X′ only at eithersaid carboxy terminus of A or at said amino terminus of A; B is apolymeric compound; X and X′ independently from each other are bivalentorganic radicals or independently from each other are present or absent;Z is a bivalent radical selected from the group consisting of: —C(R)═N—,—N═C(R)—, —CH(R)—NH—, —NH—CH(R)—, —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)—, —NH—CH(R)—Y—CH(R)—NH—, —C(R)═N—O—, —O—N═C(R)—,—CH(R)—NH—O—, —O—NH—CH(R)—, —C(R)═N—O—Y—O—N═C(R)—,—O—N═C(R)—Y—C(R)═N—O—, —CH(R)—NH—O—Y—O—NH—CH(R)— and—O—NH—CH(R)—Y—CH(R)—NH—O—; where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group and Y is abivalent organic group; wherein said conjugated derivatized protein orpolypeptide molecules of said composition are produced via formation ofa Schiff base, hydrazone, oxime or azomethine compound that results inthe presence of at least one radical having the formula —C(R)═N—, or—N═C(R)—, or, when reduced, —CH(R)—NH— or —NH—CH(R)—; and wherein saidmodified protein or polypeptide or salt thereof is prepared by a processthat employs a reaction selected from the group consisting of: reverseproteolysis, transamination, periodate oxidation and a combination orsub-combination thereof.
 43. The composition of any of claim 1, 2, 3 or42, wherein said polymeric compound B is a protein or polypeptide thatis the same or different from said protein or polypeptide A, or is areporter group or cytotoxic agent.
 44. The composition of claim 43,wherein said polymeric compound B is a reporter group comprising a metalchelating organic compound.
 45. The composition of claim 43, whereinsaid polymeric compound comprises a compound selected from the groupconsisting of: (i) desferioxamine B, or a metal derivative thereof; (ii)diethylenetriaminepentaacetic acid, or a metal derivative thereof; (iii)[Nε-(diethylenetriaminepentaacetic acid-alanyl)-Lys]₅, or a metalderivative thereof; and (iv) a polyglutamic acid having at least twoferioxamine B residues coupled thereto.
 46. The composition claim 45,wherein said reporter group is [Nε-(diethylenetriaminepentaaceticacid-alanyl)-Lys]₅ or a metal derivative thereof.
 47. A compositioncontaining modified protein or polypeptide molecules, or salts thereof,wherein said modified protein or polypeptide molecules of saidcomposition consist essentially of a compound selected from the formula: A—X—Z—X′—B wherein: A is a residue of a protein or polypeptide having acarboxy and amino terminus and is connected to X—Z—X′—B exclusively atsaid carboxy or amino terminus; B is a polymeric compound; X and X′independently from each other are bivalent organic radicals orindependently from each other are present or absent; Z is a bivalentradical selected from the group consisting of: —C(R)═N—, —N═C(R)—,—CH(R)—NH—, —NH—CH(R)—, —C(R)═N—Y—N═C(R)—, —N═C(R)—Y—C(R)═N—,—CH(R)—NH—Y—NH—CH(R)—, —NH—CH(R)—Y—CH(R)—NH—, —C(R)═N—O—, —O—N═C(R)—,—CH(R)—NH—O—, —O—NH—CH(R)—, —C(R)═N—O—Y—O—N═C(R)—,—O—N═C(R)—Y—C(R)═N—O—, —CH(R)—NH—O—Y—O—NH—CH(R)— and—O—NH—CH(R)—Y—CH(R)—NH—O—; where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group; and Y is abivalent organic group; said modified protein or polypeptide beingproduced by a method comprising condensing a compound of the formula:A—X—R¹  wherein R¹ is a —CO—R group, an acetalized formyl group, or anamino or protected amino group, and A,R, and X are as defined above,with a compound of formula: R²—X′—B  or a compound of formula: R²—Y—R² where R² is amino when R¹ is —CO—R or acetalized formyl and R2 is —CO—Ror acetalized formyl when R¹ is amino, and X′, Y, R and B are as definedabove, to form a Schiff base, hydrazone, oxime or azomethine compound,and optionally, reducing the —C(R)═N— or —N═C(R) formed by thecondensation to CH(R)—NH— or —NH—CH(R)—, respectively, and optionallyforming a salt.
 48. The composition of claim 47, wherein said polymericcompound B is a protein or polypeptide that is the same or differentfrom said protein or polypeptide A, or is a reporter group or cytotoxicagent.
 49. The composition of claim 48, wherein said polymeric compoundB is a reporter group comprising a metal chelating organic compound. 50.A composition containing conjugated protein or polypeptide derivatives,wherein said conjugated protein or polypeptide derivatives of saidcomposition have a C-terminus and an N-terminus and consist essentiallyof a compound selected from the group consisting of (1) a protein orpolypeptide that is connected only at said C-terminus to a reportergroup through a Schiff-base radical of the formula —C(R)═N— or —N═C(R)—,or, when reduced, a radical of the formula —CH(R)—NH— or —NH—C(R)—, and(2) a protein or polypeptide that is connected only at said N-terminusto a reporter group through a Schiff-base radical of the formula—C(R)═N— or —N═C(R)—, or, when reduced, a radical of the formula—CH(R)—NH— or —NH—C(R)—, where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group.
 51. Thecomposition of claim 50, wherein said conjugated protein or polypeptidederivatives of said composition consist essentially of protein orpolypeptide derivatives that are conjugated exclusively at theirC-terminus to said reporter group.
 52. The composition of claim 50,wherein said conjugated protein or polypeptide derivatives of saidcomposition consist essentially of protein or polypeptide derivativesthat are conjugated exclusively at their N-terminus to said reportergroup.
 53. The composition of claim 51 or 52, wherein said reportergroup is selected from the group consisting of a metal chelator andpolymeric compound.
 54. The composition of claim 53, wherein saidreporter group comprises a polymeric compound.
 55. The composition ofclaim 54, wherein said polymeric compound comprises a protein orpolypeptide.
 56. A composition containing conjugated protein orpolypeptide derivatives, wherein said conjugated protein or polypeptidederivatives of said composition have a C-terminus and an N-terminus andconsist essentially of a compound selected from the group consisting of(1) a protein or polypeptide that is connected only at said C-terminusto a polymeric compound through a Schiff-base radical of the formula—C(R)═N— or —N═C(R)—, or, when reduced, a radical of the formula—CH(R)—NH— or —NH—C(R)—, and (2) a protein or polypeptide that isconnected only at said N-terminus to a polymeric compound through aSchiff-base radical of the formula —C(R)═N— or —N═C(R)—, or, whenreduced, a radical of the formula —CH(R)—NH— or —NH—C(R)—, where R ishydrogen or an aliphatic, cycloaliphatic, aromatic or araliphatichydrocarbon group.
 57. The composition of claim 56, wherein said proteinor polypeptide derivatives of said composition consist essentially ofprotein or polypeptide derivatives that are conjugated exclusively attheir C-terminus to said polymeric compound.
 58. The composition ofclaim 56, wherein said protein or polypeptide derivatives of saidcomposition consist essentially of protein or polypeptide derivativesthat are conjugated exclusively at their N-terminus to said polymericcompound.
 59. The composition of claim 56 or 57, wherein said polymericcompound comprises a reporter group.
 60. The composition of claim 56 or57, wherein said polymeric compound comprises a protein or polypeptide.61. A composition containing conjugated protein or polypeptidederivatives, wherein said conjugated protein or polypeptide derivativesof said composition have a C-terminus and an N-terminus and consistessentially of a compound selected from the group consisting of (1) aprotein or polypeptide that is connected only at said C-terminus to acytotoxic agent through a Schiff-base radical of the formula —C(R)═N— or—N═C(R)—, or, when reduced, radical of the formula —CH(R)—NH— or—NH—C(R)—, and (2) a protein or polypeptide that is connected only atsaid N-terminus to a cytotoxic agent through a Schiff-base radical ofthe formula —C(R)═N— or —N═C(R)—, or, when reduced, a radical of theformula —CH(R)—NH— or —NH—C(R)—, where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group.
 62. Thecomposition of claim 61, wherein said protein or polypeptide derivativesof said composition consist essentially of protein or polypeptidederivatives that are conjugated exclusively at their C-terminus to saidcytotoxic agent.
 63. The composition of claim 61, wherein said proteinor polypeptide derivatives of said composition consist essentially ofprotein or polypeptide derivatives that are conjugated exclusively attheir N-terminus to said cytotoxic agent.
 64. A composition containingconjugated protein or polypeptide derivatives, wherein said conjugatedprotein or polypeptide derivatives of said composition have a C-terminusand an N-terminus and consist essentially of a compound selected fromthe group consisting of (1) a protein or polypeptide that is connectedonly at said C-terminus to a polymeric conjugation reagent via aSchiff-base radical of the formula —C(R)═N— or —N═C(R)—, or, whenreduced, a radical of the formula —CH(R)—NH— or —NH—C(R)—, and (2) aprotein or polypeptide that is connected only at said N-terminus to apolymeric conjugation reagent through a Schiff-base radical of theformula —C(R)═NH═ or —NH—C(R)—, or, when reduced, a radical of theformula —CH(R)—NH— or —NH—C(R)—, where R is hydrogen or an aliphatic,cycloaliphatic, aromatic or araliphatic hydrocarbon group, said proteinor polypeptide derivative being produced by condensation ofcomplementary reactive moieties comprising a first reactant having amoiety selected from the group consisting of a reactive aldehyde,acetalized aldehyde and ketone, and a second reactant having a reactiveamino group, wherein at least one of said reactants is a protein orpolypeptide having a C- or N-terminus comprising said aldehyde,acetalized aldehyde, ketone or amino group.
 65. The composition of claim64, wherein said protein or polypeptide derivatives of said compositionconsist essentially of protein or polypeptide derivatives that areconjugated exclusively at their C-terminus to said conjugation reagent.66. The composition of claim 64, wherein said protein or polypeptidederivatives of said composition consist essentially of protein orpolypeptide derivatives that are conjugated exclusively at theirN-terminus to said conjugation reagent.
 67. The composition of any ofclaim 65 or 66, wherein said conjugation reagent comprises a reportergroup.
 68. The composition of any of claim 65 or 66, wherein saidconjugation reagent comprises a protein or polypeptide.
 69. Thecomposition of any of claim 65 or 66, wherein said conjugation reagentcomprises a cytotoxic agent.